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Immunology

Synthetic CD4+ T Cell–Targeted Antigen-Presenting Cells Elicit Protective Antitumor Responses

Stefano Caserta, Patrizia Alessi, Jlenia Guarnerio, Veronica Basso and Anna Mondino
Stefano Caserta
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Patrizia Alessi
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Jlenia Guarnerio
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Veronica Basso
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Anna Mondino
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DOI: 10.1158/0008-5472.CAN-07-5796 Published April 2008
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Abstract

CD4+ helper T cells are critical for protective immune responses and yet suboptimally primed in response to tumors. Cell-based vaccination strategies are under evaluation in clinical trials but limited by the need to derive antigen-presenting cells (APC) from patients or compatible healthy donors. To overcome these limitations, we developed CD4+ T cell–targeted synthetic microbead-based artificial APC (aAPC) and used them to activate CD4+ T lymphocytes specific for a tumor-associated model antigen (Ag) directly from the naive repertoire. In vitro, aAPC specifically primed Ag-specific CD4+ T cells that were activated to express high levels of CD44, produced mainly interleukin 2, and could differentiate into Th1-ike or Th2-like cells in combination with polarizing cytokines. I.v. administration of aAPC led to Ag-specific CD4+ T-cell activation and proliferation in secondary lymphoid organs, conferred partial protection against subcutaneous tumors, and prevented the establishment of lung metastasis. Taken together, our data support the use of cell-free, synthetic aAPC as a specific and versatile alternative to expand peptide-specific CD4+ T cells in adoptive and active immunotherapy. [Cancer Res 2008;68(8):3010–8]

  • Helper T cells
  • Antigen presentation
  • Cell proliferation
  • Immunotherapy

Introduction

CD4+ T-cell help is critical for the development and the maintenance of protective immune responses. Studies on mouse models of tumor disease ( 1– 3) and cancer patients ( 4– 7) have revealed that although tumor-specific CD4+ T-cell responses do arise naturally, various degrees of tolerance eventually develop ( 3, 8– 10). CD4+ T cell–directed vaccination has been attempted to promote responses and in some instances shown to augment antitumor responses ( 11– 16). However, cell-based vaccination strategies are currently limited by the need to derive autologous ( 17) and heterologous ( 18) antigen-presenting cells (APC) through lengthy good manufacturing procedures.

To overcome such limitations, synthetic cell-free artificial APC (aAPC) have been developed. These include microbeads coated with anti-CD3/CD28 stimulatory antibodies able to elicit polyclonal T-cell expansion of short-lived effector lymphocytes ( 19) or with whole tumor cell membranes (large multivalent immunogens) capable of eliciting tumor-specific effector CD8+ T-cell responses in vitro ( 20, 21). As such, large multivalent immunogens showed some efficacy in a human phase I trial ( 22). After the identification of several MHC class I–restricted tumor-associated antigens (Ag) and the availability of recombinant MHC-I molecules, Ag-specific CD8+ T cell–targeted aAPC were also developed by immobilizing MHC-I/peptide complexes on microbeads ( 20, 21, 23, 24) and liposomes ( 25, 26). Such aAPC induced specific CD8+ T-cell activation and proliferation in vitro and showed antitumor potential in vivo ( 27– 29).

Fewer attempts have been made to target the aAPC strategy to CD4+ T cells in vitro ( 24, 30), and no report exists about their applications in vivo. In recent studies, peptide-HLA–coated beads were used in vitro to expand influenza-specific CD4+ T lymphocytes from peripheral blood lymphocytes ( 24, 30), and the expansion was dependent on the presence of additional feeder cells, suggesting the need for costimulation. In the same study, virus-specific CD4+ T cells could be expanded from infected individuals and not healthy subjects after polyclonal activation and tetramer-specific sorting of memory CD4+ T cells ( 24, 30).

Thus, whether MHC-II–based aAPC might be sufficient to prime Ag-specific CD4+ T cells directly from a naive repertoire and to improve antitumor immunity in vivo remains to be determined. To address these issues, we developed synthetic microbead-based aAPC coated with peptide–MHC-II dimers [I-Ad/Leishmania receptor for activated C kinase (LACK); ref. 31] and anti-CD28 stimulating monoclonal antibody (mAb; L/28 aAPC). We report the ability of CD4+ T cell–directed aAPC to induce activation and proliferation of Ag-specific naive CD4+ T cells in primary T-cell cultures in vitro and in secondary lymphoid organs in vivo and to sustain protective responses against subcutaneous and lung tumors.

Materials and Methods

Proteins and aAPC preparation. I-Ad/LACK molecules consist of recombinant I-Ad–Fc fusion proteins covalently attached to immunodominant peptide from the Leishmania LACK antigen ( 31). Dimers were obtained from stably transfected Drosophila melanogaster cells and purified by protein G affinity chromatography and ionic exchange fast-protein liquid chromatography (Amersham Biotech) as previously described ( 31). aAPC were prepared by incubating 5 μm polystyrene-sulfate latex microparticles (Invitrogen; 20 × 106/mL) in PBS containing different amounts of the proteins of interest: I-Ad/LACK dimers (0–100 μg/mL) or commercially available anti-CD3 (clone 145-2C11, eBioscience; 0.05 μg/mL) and/or anti-CD28 antibodies (clone 37.51, eBioscience; 2 μg/mL). Coating was allowed to occur for 20 min at 4°C on a rotating wheel. Microparticles were then washed twice in PBS with 2% FCS, and free sites were blocked by 30-min incubation at room temperature in complete RPMI with 5% FCS. The following aAPC were prepared: I-Ad/LACK dimers and anti-CD28 mAb (referred as L/28 aAPC), anti-CD3 and anti-CD28 mAb (referred as 3/28 aAPC), and anti-CD28 mAb only (referred as -/28 aAPC). Adsorption of the proteins onto the microparticle surface was monitored by flow cytometry by staining with a FITC-labeled goat anti-hamster mAb (Caltag Laboratories) and a PE-labeled anti–I-Ed/I-Ad mAb (BD Biosciences), which recognize the anti-CD28 mAb and I-Ad/LACK dimers, respectively.

Mice, tumor cells, in vivo immunization protocol, and BrdUrd administration. Seven-week-old to 8-week-old BALB/c mice were purchased from Charles River (Charles River Italia). DO11.10 ( 32) and 16.2β Transgenic (Tg; ref. 31) mice were bred in the specific pathogen–free facility of the institute. T lymphocytes derived from 16.2β mice express a Tg TCR β-chain specific for an I-Ad–restricted peptide (LACKp, FSPSLEHPIVVSGSWD) derived from for the Leishmania major–derived Ag LACK. As a result of pairing with endogenous TCR α-chains, only ∼0.5% of CD4 T cells in 16.2β Tg animals are LACK-specific and can be detected with I-Ad/LACK specific multimers (see below). T lymphocytes derived from DO11.10 mice express a Tg αβ-TCR specific for an I-Ad–restricted peptide (OVAp, ISQAVHAAHAEINEAGR) derived from chicken ovalbumin. As a result, 100% of CD4+ T cells are ovalbumin-specific.

TS/A-LACK tumor cells were previously described ( 3). Briefly, TS/A cells were transfected with a pcDNA3-derived vector encoding for a truncated form of the L. major-derived model Ag LACK and selected by limiting dilution of G-418–resistant clones ( 3). Tumor cells were grown in vitro at 37°C in complete medium (RPMI 1640 with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 units/mL streptomycin, and 2.5 × 10−5 mol/L 2-ME, G-418 0.5 mg/mL; Invitrogen Life Technologies). In these cells, which do not express MHC-II molecules, LACK is expressed as a cytosolic protein and is representative of a tumor-associated Ag ( 3). Exponentially growing TS/A-LACK tumor cells were either s.c. (4 × 105) or i.v. (2 × 105) injected in 100 μL of PBS. The development of subcutaneous tumors was monitored over time as previously described ( 3). Lungs were recovered 20 d after tumor cell injection, and metastatic foci were dissected and counted.

Where indicated, mice were immunized i.v. with 20 × 106 aAPC resuspended in 100 μL of PBS 20 d before tumor challenge, and boosted 10 d later. As control, mice were immunized s.c. with 2 × 105 lipopolysaccharide (LPS)–matured bone marrow–derived peptide-pulsed dendritic cells (DC/LACK) prepared as previously described ( 3, 33). Briefly, bone marrow precursors were propagated for 7 d in complete Iscove's medium containing 25 ng/mL mouse rGM-CSF and 5 ng/mL mouse rIL-4 (Peprotech). Bone marrow–derived DCs were then allowed to mature in the presence of LPS (1 μg/mL; Sigma-Aldrich) for 8 h and pulsed for 1 h with 2 μmol/L class II–restricted immunodominant peptide LACK. DC maturation and purity were routinely evaluated by flow cytometry after staining with mAb recognizing CD11c, MHC class II, B7.1, B7.2, and CD40 molecules (all from BD Biosciences).

In selected experiments, BrdUrd (Sigma) was injected in the peritoneal cavity at time 0 (4 mg/mL, 300 μL of PBS) and provided in the drinking water (0.8 mg/mL) for the length of the experiment.

Five to 10 mice per group were used in each experiment. All the in vivo studies were approved by the Ethical Committee of the San Raffaele Scientific Institute and done according to its guidelines.

T-cell primary cultures. Mice were sacrificed and the axillary, brachial, and inguinal lymph nodes (LN) and the spleen were surgically excised. Organs were forced through a nylon screen to make single-cell suspensions, and cells were washed and resuspended in complete medium (RPMI with 5% FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 2.5 × 10−5 mol/L 2-ME; Invitrogen Life Technology). Blood was obtained by intracardiac puncture and immediately mixed with heparin (Sigma). Thereafter, blood cells were washed and resuspended in PBS and layered onto Lympholyte-M (2:1 ratio, Cedarlane) to separate mononuclear cells by centrifuging at 2,000 × g for 30 min at room temperature. The lymphocyte layer was then rescued and washed twice in PBS.

2 × 106 LN, 3 × 106 spleen, or 5 × 106 blood-derived cells were plated (24-well plate, 1 mL) with 2.5 × 106 aAPC or alternatively with the same number of irradiated syngeneic splenocytes in the presence of the I-Ad restricted peptides: OVA323-339 (0.25 μmol/L) or LACK158-173 (5 μmol/L). Where specified, cells were cultured with aAPC under Th1 or Th2 polarizing conditions respectively by the addition of murine interleukin 12 (IL-12; 5 ng/mL, Peprotech) and anti–IL-4 neutralizing mAb (2.5 μg/mL, clone 11B11, BD PharMingen) or murine IL-4 (5 ng/mL, Peprotech) and anti-IL-12 neutralizing mAb (2.5 μg/mL, clone C17.8, BD PharMingen). In addition, aAPC were combined with murine IL-7 (100 ng/mL, Peprotech).

In selected experiments, cells were labeled with the vital dye, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Invitrogen), final concentration of 1 μmol/L. Briefly, cells were washed twice with PBS and suspended at 20 × 106/mL in PBS. An equal volume of CFSE at 2 μmol/L was then added. After an 8-min incubation under gentle shaking at room temperature, an equal volume of FCS was added to quench the reaction. Thereafter, the cells were washed twice in RPMI with 10% FCS and plated with aAPC as described above. T-cell proliferation was independently evaluated by [3H]yhymidine incorporation. Briefly, 2 × 105 LN cells were cultured with 2.5 × 105 aAPC in 96-well plate (200 μL complete medium). After 48 h, the cultures were pulsed with [3H]thymidine (1 μCi/well) and harvested after an additional 18 h.

I-Ad/LACK multimer staining and BrdUrd detection. I-Ad/LACK multimer staining was done as previously described ( 3). Briefly, I-Ad/LACK multimers were obtained by incubating I-Ad/LACK dimers (3 μg/sample) with Alexa 488–coupled protein A (Molecular Probes, Invitrogen; 0.3 μg/sample) in PBS for 30 min at RT. Free protein A–binding sites were saturated by the addition of total IgG (1 μg/sample). LN cells (6 × 105) were first incubated with a blocking buffer (5% rat serum and 95% culture supernatant of 2.4G2 anti-FcR mAb-producing hybridoma cells) for 20 min to saturate the FcRs and then stained with I-Ad/LACK multimers for 1 h on ice in PBS supplemented with 0.5% bovine serum albumin. Thereafter, the cells were stained with PE-labeled or PerCP-labeled anti-CD4, anti-CD69, anti-CD44, and anti-CD62L mAb and with allophycocyanin-labeled anti-CD8a, anti-CD11b, and anti-B220 mAb (BD PharMingen). TOPRO-3 (1 nmol/L final concentration; Molecular Probes, Invitrogen) was added to the sample just before flow cytometric analysis to discriminate viable and dead cells. CD8a+CD11b+B220+TOPRO+ cells were excluded by electronic gating during the acquisition. Fifty to 100 × 103 CD4+ T cells were acquired using a FACSCalibur flow cytometer (BD Biosciences).

BrdUrd incorporation was determined as previously described ( 34) by flow cytometry. In particular, after 3 d from aAPC immunization, mice were killed and cells were derived from lymphoid organs recovered as described above. Typically, 1 to 2 × 106 cells were initially surface-stained with PerCP-labeled anti-CD4, PE-labeled anti-CD44, and APC-labeled anti-CD8a, anti-CD11b, and anti-B220 mAb (BD PharMingen). The cells were then fixed with 2% ice-cold formaldehyde, overnight at 4°C. The following day, cells were washed in PBS, permeabilized for 2 to 3 min at 4°C [0.1% Triton-X-100, 0.1% Na-Cytrate (pH 7.2)], washed again in PBS and then in 40 mmol/L Tris-HCl, 10 mmol/L NaCl, and 6 mmol/L MgC12 (pH 8.0). Thereafter, the cells were incubated for 1 h at 37°C in the same buffer containing 50 Kunitz units of DNase I (Sigma). After washing in PBS/0.5% Tween 20, FITC-labeled anti-BrdUrd or hamster-IgG-isotype control antibody (BD Biosciences) were added 30 min at RT, washed in PBS/0.5% Tween 20, and washed one last time in PBS. Fifty to 100 × 103 CD4+CD8a−CD11b−B220− events were collected in the viable gate. The background levels in the staining were measured for each singular sample with the hamster-IgG-isotype–stained control and subtracted in the absolute number calculations.

Cytokine secretion assays and ELISA. For intracellular cytokine detection, 5 × 105 cells were stimulated with 5 × 106 L/28 aAPC (I-Ad/LACK dimers, 20 μg/mL; anti-CD28 mAb, 2 μg/mL) or -/28 aAPC for 5 h at 37°C, of which the last two are in the presence of Brefeldin A (5 μg/mL, Sigma). Thereafter, the cells were surface-stained with PerCP-labeled anti-CD4 mAb, fixed in 2% formaldehyde, and permeabilized in PBS containing 2% FCS, 0.5% saponin, 2% rat serum, and 0.2% sodium azide (permeabilization buffer). The cells were then stained with anti–IL-2, anti–IL-4, anti–IFN-γ, anti–IL-10, anti–IL-17 mAb in permeabilization buffer for 30 min at RT in the darkness. Finally, the cells were washed in permeabilization buffer and then in PBS with 2% FCS. Fifty to 100 × 103 CD4+ events were generally collected in the viable gate on a FACSCalibur. Cytokine release induced by L/28 aAPC was comparable with the one induced by LACK-pulsed syngeneic splenocytes (not shown).

In selected experiments, LN-derived cells (2 × 105) were stimulated with the LACK158-173 peptide (0–10 μmol/L) and irradiated syngeneic splenocytes (6 × 105, 5,000 rad) in 96-well plate. IL-2 and IFN-γ were measured in 24-h culture supernatants by capture ELISA according to the protocol provided by the manufacturer (BD PharMingen).

Statistical analysis. Statistical analyses were done using unpaired two-tailed Student's t test.

Results and Discussion

Synthetic aAPCs suitable to prime antigen (Ag)–specific CD4+ T cells were developed by decorating polystyrene sulfate microspheres (5-μm diameter) with purified peptide–MHC-II dimers (I-Ad/LACK; ref. 31) and anti-CD28 stimulating mAb (clone 37.51; referred to as L/28 aAPC). Such aAPC should thus provide the two major signals needed for T-cell activation: MHC/TCR stimulation together with CD28 costimulation. The amount of Ag immobilized on the beads was controlled titrating the I-Ad/LACK dimers (0–100 μg/mL) used to coat a fixed number of beads. A fixed amount of anti-CD28 antibody (2 μg/mL) was used at the same time. Homogeneous protein adsorption on the beads was confirmed by flow cytometry analyses after staining with specific antibody to detect I-Ad/LACK and anti-CD28 mAb, respectively ( Fig. 1A , representative dot plots). Binding of I-Ad/LACK dimers increased up to coating concentration of 40 μg/mL and reached a plateau at higher concentration ( Fig. 1A, squares in graph). Coating of the anti-CD28 mAb was optimal in the presence of 5 to 20 μg/mL I-Ad/LACK dimers ( Fig. 1A, diamonds in graph).

Figure 1.
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Figure 1.

Development and validation of synthetic CD4+ T cell–targeted aAPC. A, 5-μm polystyrene sulfate microparticles were incubated with variable amounts of purified I-Ad/LACK dimers and optimized amount (2 μg/mL) of anti-CD28 mAb (clone 37.51). Immobilization of the proteins onto the bead surface was analyzed by flow cytometry by staining of the microparticles with a FITC-labeled anti-hamster antibody and a PE-labeled anti-MHCII mAb, which reveal the anti-CD28 and I-Ad/LACK dimer, respectively. Dot plots compare an uncoated bead sample to a representative preparation of microspheres. The mean fluorescence intensity of I-Ad/LACK dimers (squares) and anti-CD28 mAb (diamonds) immobilized on the aAPC is depicted in the graph (right). B, C, and D, L/28 aAPC were used to stimulate CFSE-labeled 16.2β (left) and DO11.10 (right) LN cells (2.5:1 ratio). Cells were also stimulated with irradiated syngeneic splenocytes (as APC) and nominal Ag (Ag): LACKp (5 μmol/L; left) and OVAp (0.25 μmol/L, right) respectively. After 5 d of culture, cells were stained with anti-CD4 mAb and TO-PRO-3 to exclude dead cells, and the CFSE content of CD4+ viable (TO-PRO-3−) T cells was determined by fluorescence-activated cell sorting analyses and depicted in the histograms in B. In C, the percentage of viable CD4+ CFSEdim T cells proliferating after stimulation with L/28 aAPC bearing different concentrations of I-Ad/LACK dimers and anti-CD28 (shown in A) is reported. [3H]thymidine was added to a set of culture, and incorporation was measured after an additional 18 h (C, inset). D, 16.2βCD4+ T cells were purified (94% pure), labeled with CFSE, and cultured for 5 d with -/28 APC or L-28 aAPC. The relative CFSE content is depicted.

The ability of L/28 aAPC to support Ag-specific naive T-cell proliferation was determined by the CFSE dilution assay ( 35). We compared CD4+ T-cell proliferation induced in cultures derived from 16.2β Tg mice (in which ∼0.5% of CD4+ T cells expresses a LACK-specific TCR; see Materials and Methods; ref. 31) and DO11.10 Tg mice (in which all the T cells express an ovalbumin-specific I-Ad–restricted TCR; ref. 32). LN-derived cells were stained with the CFSE vital dye and cultured with L/28 aAPC, and after 5 days CD4+ T-cell proliferation was revealed by the appearance of cells with a dimmer CFSE profile. CD4+ T-cell proliferation was detectable in cultures of 16.2β Tg mice ( Fig. 1B, middle) and was comparable with that induced by the nominal Ag (LACKp; Fig. 1B, bottom). DO11.10-derived CD4+ T cells did not expand in response to L/28 aAPC ( Fig. 1B, middle) but proliferated to the nominal Ag (OVAp; Fig. 1B, bottom). Thus, L/28 aAPC support the expansion of naive CD4+ T lymphocytes, and this is Ag-specific.

LACK-specific CD4+ T-cell proliferation, both determined by CFSE dilution (day 5; Fig. 1C) or [3H]thymidine incorporation (48 h; Fig. 1C, inset), was maximal in response to L/28 aAPC bearing optimal amounts of I-Ad/LACK dimers and anti-CD28 mAb (5–20 and 2 μg/mL, respectively; Fig. 1C compare with A). Of note, the coimmobilization of anti-CD28 mAb allowed to reduce considerably the amount of I-Ad/LACK dimers needed to elicit optimal T-cell proliferation.

The LACK peptide is covalently linked to the MHC β-chain of I-Ad/LACK dimers and accommodated within the Ag-binding groove in the MHC-II molecules ( 31). Nevertheless, to exclude the possibility that the peptide might be shed from the recombinant molecule and represented by the endogenous APC and verify that L/28 aAPC could directly present Ag to naive CD4+ T cells, cultures were set up with highly purified 16.2β CD4+ T lymphocytes. Also in this case, L/28 aAPC, but not -/28 APC, elicited the proliferation of a fraction of the CD4+ T cell, supporting the ability of peptide/MHC class II tetramers/anti-CD28 mAb aAPC to activate naive T cells ( Fig. 1D).

L/28 aAPC-driven T-cell expansion was accompanied by the enrichment of LACK-specific CD4+ T cells also enumerated by flow cytometry analyses using I-Ad/LACK multimer staining ( 3). I-Ad/LACK+ CD4+ T cells increased both in frequency ( Fig. 2A ) and in total number ( Fig. 2B) in L/28 aAPC or LACK peptide–driven 16.2β, but not DO11.10 LN-derived cultures at day 7 (compare white and black bars in Fig. 2B). In control -/28 and 3/28 aAPC-driven cultures, the frequency (not shown for 3/28 aAPC) and the absolute numbers of LACK-specific CD4+ T cells remained comparable with that found ex vivo ( Fig. 2A and B). Peptide stimulation in the presence of irradiated splenocytes as APC led to better yields of Ag-specific CD4+ T cells ( Fig. 2B), possibly reflecting the contribution of other accessory molecules, which can be found on the surface of such APC. However, as reported for Flu-specific Ag-primed CD4+ T cells ( 30), CD4+ T lymphocytes expanded by the nominal Ag presented by feeder cells were less prone to subsequent Ag-driven restimulations than aAPC expanded cells. 3 Thus, L/28 aAPC seems to be an effective and defined tool to increase LACK-specific CD4+ T cells by several times starting from a naive repertoire.

Figure 2.
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Figure 2.

Synthetic L/28 aAPC elicit antigen-specific CD4+ T-cell expansion. 16.2β (A, B, and D), DO11.10 (B and C), and BALB/c (C) cells derived from LN (A and B) and blood (C and D) were analyzed ex vivo and/or after an in vitro culture with -/28 aAPC, L/28 aAPC, and 3/28 aAPC or with LACK-pulsed irradiated splenocytes (LACKp) by flow cytometry after staining with anti-CD4, anti-B220, anti-CD11b, anti-CD8 mAbs, I-Ad/LACK multimers, and TO-PRO-3 dye to exclude dead cells. A, viable TO-PRO-3−B220−CD11b−CD8−CD4+ cells are depicted in the plots. The frequency of I-Ad/LACK+ T cells is reported. B, the bar graph depicts the total number of TO-PRO-3−B220−CD11b−CD8−CD4+ I-Ad/LACK+ T cells in 16.2β Tg (black columns)– and DO11.10 (white columns)-derived cultures at day 7. C, DO11.10 (top) and BALB/c (bottom) lymphocytes derived from blood were cultured with L/28 aAPC. Panels show viable TO-PRO-3−B220−CD11b−CD8−CD4+ T cells. Ag-specific I-Ad/LACK+ CD4+ T cells were analyzed for CD44 expression, and percentages are reported in each panel. D, 16.2β-derived PBMC were labeled with the CFSE vital dye and cultured for a week with control, -/28 aAPC, or L/28 aAPC, and analyzed by flow cytometry for CFSE content after staining with anti-CD4, anti-CD44. and anti-CD62L mAbs at day 7. Events are shown after gating on viable CD4+ T cells.

It was possible that this phenomenon was a particular feature of the Tg model used here, having a higher frequency of LACK-specific naive precursors. To answer this question, we did similar experiments using peripheral blood mononuclear cells (PBMC) of BALB/c mice, which have a normal T-cell repertoire. LACK-specific CD4+ T cells accumulated to sizable numbers in naive BALB/c blood-derived cultures ( Fig. 2C, bottom), whereas remained undetectable in control DO11.10 (ovalbumin restricted) cultures ( Fig. 2C, top). Altogether, these data indicate that L/28 aAPC can prime and expand LACK-restricted CD4+ T cells from both 16.2β Tg and normal (BALB/c) naive repertoires.

The surface and functional phenotype of aAPC-primed CD4+ T cells was next investigated. PBMCs of 16.2β mice were CFSE labeled and cultured for 6 days with -/28 aAPC or L/28 aAPC and analyzed by flow cytometry ( Fig. 2D) for the expression of activation markers. As expected, CD4+ T cells cultured with -/28 aAPC retained the original CFSE content, did not increase in size, and revealed CD4, CD44, CD62L, and CD25 (not shown) surface levels comparable with those found on naive animals ex vivo. In L/28 aAPC-driven cultures, a fraction of CD4+ T cells diluted its CFSE content, increased in size, and up-regulated the expression of CD4, CD44 ( Fig. 2D), CD69, and CD25 (not shown). Proliferating cells were distributed among CD62Lhigh and CD62Llow cells ( Fig. 2D), as also in the case of LACK peptide–primed lymphocytes (not shown).

The ability of cultured cells to produce cytokines was analyzed after control [-/28 or phorbol 12-myristate 13-acetate (PMA) and Ionomycin] or LACK-specific restimulation (L/28; Fig. 3 ). Upon control (-/28) restimulation, negligible amounts of cytokine secretion were found ( Fig. 3A). At difference, upon LACK-specific stimulation (L/28), a sizable frequency of CD4+ T cells in L/28 aAPC-driven and LACK peptide–driven cultures, but not in control -/28 aAPC–driven cultures, expressed IL-2 ( Fig. 3A). A small fraction of IL-2+/IFN-γ+ and IFN-γ+ CD4+ T cells was also found, whereas IL-4 secretion remained within background levels ( Fig. 3A and B). Thus, aAPC induce activation and proliferation of Ag-specific CD4+ T cells, without favoring their polarization. Nonetheless, as it might be relevant for both adoptive and active immunotherapy applications to generate Ag-specific CD4+ T cells with different helper capability, we investigated whether it was possible to combine L/28 aAPC with recombinant cytokines and favor CD4+ helper T-cell polarization. Again, in the presence of the L/28 aAPC alone, most of the LACK-specific CD4+ T cells produced only IL-2 upon LACK-specific stimulation (∼11% in Fig. 3B). However, when cultures were done with L/28 aAPC and recombinant IL-12 (Th1 polarization), LACK-specific IFN-γ–producing CD4+ T cells were instead induced (∼11%; Fig. 3B). Conversely, culturing the cells with L/28 aAPC and recombinant IL-4 (Th2 polarization) favored the differentiation of LACK-specific IL-4 producing CD4+ T cells ( Fig. 3B). This suggests that L/28-driven T-cell activation does not drive terminal differentiation of activated cells and rather preserve the lymphocyte susceptibility to exogenous cytokines, which drive their differentiation into Th1-like or Th2-like effectors. In addition, L/28 aAPC were combined with IL-7, a known survival factor for T cells ( 36), the accumulation of multifunctional IL-2+, IL-2+/IFN-γ+, and IFN-γ+ IL-4− CD4+ T cells ( Fig. 3B). Of note, up to 50% of L/28 and L/28+IL-7 cultured CD4+ T cells expressing high levels of CD44 maintained the surface expression of the LN homing molecule CD62L ( Fig. 3C).

Figure 3.
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Figure 3.

L/28 aAPC expand Ag-specific polarized CD4+ T-cell subsets in combination with cytokines. A, 16.2β LN cells were cultured with -/28 aAPC, L/28 aAPC, or LACK-pulsed irradiated splenocytes for 7 d as indicated in the figure. Thereafter, cultures were harvested and cells were stimulated for 5 h with -/28 aAPC (-), L/28 aAPC, or with PMA and ionomycin (P/I). Cells were then stained with anti-CD4 mAb, fixed, permeabilized, and stained with anti–IL-2, anti–IFN-γ, and anti–IL-4 mAbs to measure cytokine content in intracellular analysis. The frequency of viable CD4+ cytokine-producing cells is depicted. Control (-; P/I) stimulation is depicted for L/28 aAPC cultured cells, but comparable results were obtained in the other experimental conditions (not shown). B, 16.2β LN cells were cultured with L/28 aAPC in the absence (L/28 aAPC) or in the presence of recombinant Th1 or Th2 polarizing cytokines (L/28 Th1, IL-12 and anti–IL-4 blocking mAb; L/28 Th2, IL-4 and anti–IL-12 blocking mAb) or with IL-7 (L/28 + IL-7). On day 7, the cells were analyzed for cytokine secretion capability after control (-/28) and LACK-specific (L/28) 5-h stimulation. The frequency of viable CD4+ cytokine producing cells is depicted. Control (-/28) stimulation is depicted for L/28+IL-7 aAPC cultured cells. With the exception of cells cultured in Th2 polarizing condition, IL-4 expression remained within background levels (B, bottom row). C, the relative surface expression of CD44 and CD62L on cells cultured with IL-7, L/28 aAPC, or L/28 aAPC and IL-7 in combination (L/28 + IL-7) were analyzed by flow cytometry after staining with I-Ad/LACK multimers. Events are shown after gating on viable TO-PRO-3−B220−CD11b−CD8−CD4+ I-Ad/LACK+ T cells. The experiments are representative of three to five independent determinations.

Taken together, the data indicate that synthetic peptide-MHC class II and anti-CD28 mAb-coated aAPC elicit the Ag-specific activation and expansion of CD4+ T cells from a naive normal T-cell repertoire and in combination with recombinant cytokines can be used to differentiate effector lymphocytes in vitro. Such versatility of aAPC might be particularly relevant in adoptive immunotherapy applications whenever multifunctional memory-like CD4+ T cells or unpolarized CD4+ T cells, which might have a superior therapeutic activity when compared with terminally differentiated effector T cells ( 37– 39) are needed.

We next investigated whether L/28 aAPC could elicit CD4+ T-cell responses in vivo in 16.2β Tg mice. This model system was previously exploited to trace L. major ( 31), tumor ( 3), and DC vaccine–induced CD4+ T-cell responses ( 10). Control, -/28, and L/28 aAPC (2 × 107) were injected in the tail vein. BrdUrd was used to evaluate in vivo cell proliferation. LACK-pulsed bone marrow–derived mature DCs ( 3, 10) were injected into a separate group of mice to compare aAPC to professional APC. AAPC i.v. administration resulted in systemic redistribution as evidenced by beads accumulation in well-vascularized organs, such as the spleen and the lungs, but did not affect the health of the animals for the time of observation (not shown). Three days after injection, a group of mice was sacrificed and cells derived from the peripheral LN (axillary, brachial, and cervical) were analyzed by flow cytometry after staining with the I-Ad/LACK+ multimers, anti-CD4, anti-CD44, anti-CD69, and anti-CD62L ( Fig. 4 ). At day 3, the frequency of I-Ad/LACK+ CD4+ T cells increased in the peripheral LN of mice immunized with L/28 aAPC, whereas in control, mice immunized with -/28 aAPC remained comparable with the one found in naive mice ( Fig. 4A). Furthermore, a fraction of I-Ad/LACK+ CD4+ T cells in L/28 aAPC immunized mice increased in size, expressed high levels of CD69, CD44 ( Fig. 4B), and CD25 (not shown), and had down-regulated the surface expression of CD62L ( Fig. 4B). At day 3, L/28 aAPC-mediated CD4+ T-cell activation was comparable with that induced by LACK-loaded DC ( Fig. 4A and B). By this time, L/28 and not -/28 aAPC immunized mice also showed a higher frequency of CD4+ CD44high T cells, which had incorporated BrdUrd after immunization ( Fig. 4C). These data strongly suggest that Ag-specific CD4+ T cells were activated (CD44high) and divided (BrdUrd+) in L/28 aAPC immunized mice. Ten days after L/28 aAPC injection, the frequency of I-Ad/LACK+ CD4+ T cells had returned to baseline levels and was comparable with that found in control mice. However, a fraction of LACK-specific CD4+ T cells expressed high levels of CD44 indicative of previous Ag encounter ( Fig. 4D). To analyze the Ag-specific cytokine secretion profile, LN cells were stimulated with LACK peptide for 24 hours and cytokines were measured in culture supernatants by ELISA. LACK-specific IL-2 release was detectable in LN cultures of L/28 aAPC-LACK ( Fig. 5A) and DC-LACK day 3 immunized mice ( 3, 10), but not in those of naive and -/28 aAPC immunized mice ( Fig. 5A ). Similarly to what we found in vitro ( Fig. 3A and B), LACK-specific IFN-γ secretion was not found in culture supernatants of L/28 aAPC immunized mice ( Fig. 5B). This is different from DC/LACK immunized mice, which generally show sizable frequencies of IFN-γ–producing cells ( 3, 10). IL-4, IL-10, and IL-17 remained within background detection in all samples (not shown). By day 10, LACK-specific IL-2 secreting cells were no longer detectable (not shown), possibly due to its low frequency, in spite of the presence of CD44 high cells ( Fig. 4D).

Figure 4.
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Figure 4.

L/28 aAPC activate and accumulate CD4+ T cells in secondary lymphoid organs in vivo. L/28 aAPC (2 × 107) were injected in 16.2β transgenic mice i.v. (L/28 aAPC). As control, 16.2β mice received a PBS control injection (control) or 2 × 107 anti-CD28 mAb coated aAPC (-/28 aAPC) or 2 × 105 LACK-pulsed DC s.c. (DC/LACK). BrdUrd was injected in the peritoneal cavity at time 0 and provided in the drinking water for the length of the experiment. After 3 and 10 d, a group of mice was sacrificed, and cells derived from the LN were analyzed by flow cytometry after staining with TO-PRO-3 dye and anti-CD4, anti-CD8, anti-CD11b, anti-B220, anti-CD44, anti-CD69, and anti-CD62L mAbs and I-Ad/LACK multimers (A, B, and D) or anti-CD4, anti-CD8, anti-CD11b, anti-B220, anti-CD44, and anti-BrdUrd mAb (C). Dot plot in A depicts viable TO-PRO-3−B220−CD11b−CD8− CD4+ T cells. In B, histogram overlays depict the expression of the indicated activation markers after gating on viable TO-PRO-−B220−CD11b−CD8−CD4+ I-Ad/LACK+ T cells in mice immunized with control, -/28 aAPC (gray-filled histograms), L/28 aAPC (bold black line), or DC/LACK (thin black line). In C, the frequency of BrdUrd+ CD4+ T cells is depicted in control, -/28 aAPC, and L/28 aAPC-immunized mice. Significance was evaluated with an unpaired two-tail t test. D, histograms overlay depict relative surface levels of CD44 after gating on TO-PRO-3−B220−CD11b−CD8−CD4+ I-Ad/LACK+ cells (upper) and TO-PRO-3−B220−CD11b−CD8−CD4+ I-Ad/LACK− cells (lower). Naive, -/28, and L/28 aAPC immunized mice are compared 10 d after immunization.

Figure 5.
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Figure 5.

CD4+ T cells primed by L/28 aAPC in secondary lymphoid organs mainly produce IL-2 upon restimulation. LN cells derived from L/28 or -/28 aAPC immunized mice were stimulated in vitro with increasing amounts of LACK peptide (LACKp) in the presence of irradiated syngeneic splenocytes as APC. IL-2 (A) and IFN-γ (B) production was determined in culture supernatants 24 h after stimulation by capture ELISA. Points, mean values of triplicate determination of three individual mice; bars, SD. Representative of three independent experiments done with three mice per group.

Having determined that L/28 aAPC can activate LACK-specific CD4+ T cells in vivo, we investigated whether L/28 aAPC active vaccination might confer resistance to LACK-expressing TS/A tumors ( 3). BALB/c mice were immunized i.v. with -/28 aAPC or L/28 aAPC or s.c. with LACK-pulsed DC, boosted 10 days later, and challenged s.c. with 0.4 × 106 TS/A-LACK cells. Whereas solid TS/A-LACK tumors rapidly developed in control, PBS injected (not shown), and -/28 aAPC immunized mice ( Fig. 6A ), tumor growth was delayed in L/28 aAPC and DC-LACK vaccinated mice ( Fig. 6A). By day 10, the volume of TS/A-LACK tumors in these mice was half of that found in control -/28 aAPC immunized mice ( Fig. 6A). Interestingly, depletion of CD4+ T cells after L/28 aAPC vaccination abolished the resistance to tumor growth, supporting a direct role for L/28 aAPC-activated CD4+ helper T cells in tumor protection ( Fig. 6A). By day 30, up to 40% of L/28 aAPC-immunized mice remained tumor-free (not shown) and survived up to 1 year in the absence of major clinical signs. It should be noted that, although encouraging, results obtained with L/28 aAPC were inferior to those obtained by DC-LACK immunization, which conferred long-term tumor-free survival in up to 80% of the mice ( 40).

Figure 6.
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Figure 6.

L/28 aAPC vaccination confers protection against TS/A-LACK tumors. A and B, BALB/c mice were immunized i.v. with -/28 aAPC or L/28 aAPC (20 × 106 aAPC per mouse) or s.c. with LACK-pulsed DC (DC/LACK) twice with a 10-d interval. Ten days after the last immunization, mice were challenged with 0.4 × 106 TS/A-LACK s.c. A group of mice was vaccinated and treated with CD4-depleting antibodies every 2 d, starting from 2 d before the tumor challenge (L/28 aAPC CD4 depl). Tumor growth was monitored every other day and measured with a metric caliper. A, tumor volumes at day 10 are depicted. Statistical relevance was determined by two-tailed t test. B and C, BALB/c mice (7 mice per group) were immunized with -/28 aAPC or L/28 aAPC i.v. (20 × 106 aAPC per mouse) or with DC/LACK (DC/LACK) s.c. and boosted after 10 d. A day later, mice were challenged i.v. with 0.2 × 106 TS/A-LACK cells. Lung metastases were counted 25 d after challenge. In B, representative lungs are depicted. In C, the mean number of metastases per lung is reported. Statistical relevance was determined by two-tailed t test; *, P < 0.05; **, P < 0.01; ***, P < 0.0002; ns, not statistically different. PBS and -/28 aAPC mice were not statistically different (P = 0.07).

As we found that aAPC localized also in the lungs after i.v. administration (not shown), we asked whether L/28 aAPC could protect against lung tumor metastasis. BALB/c mice were left untreated or immunized i.v. with -/28 aAPC or L/28 aAPC or s.c. with DC-LACK, boosted 10 days later, and challenged i.v. with 2 × 105 TS/A-LACK cells. After i.v. injection TS/A-LACK tumor cells rapidly generated lung metastases in control (PBS) and -/28 aAPC treated mice ( Fig. 6B and C). In contrast, both L/28 aAPC and DC-LACK immunized mice proved resistant to TS/A-LACK lung tumor metastasis to a comparable extent ( Fig. 6B and C). Together, these data indicate that CD4+ T-cell targeted aAPC induce specific CD4+ T-cell activation and proliferation in vivo and support antitumor responses.

Concluding Remarks

We report here that latex bead–based aAPC presenting a single MHC class II peptide derived from a tumor-associated model antigen (LACK) in the context of CD28-mediated costimulation is able to activate Ag-specific CD4+ T cells, in vitro and in vivo, and also confer resistance to subcutaneous tumors and lung metastasis. Although less potent than bone marrow–derived DC, synthetic MHC-II/peptide-based aAPC might provide a suitable alternative to favor helper functions and the development of protective immune responses in vitro and in vivo. Such aAPC might provide the opportunity to bypass the need to derive autologous and heterologous APC under good manufacturing procedure conditions in clinic and obtain defined APC preparation (i.e., optimal Ag densities and costimulatory abilities) suitable for large-scale production. Latex-based microspheres had no significant side effects when given i.v. in mice ( 27, 29) and s.c. or i.d. in humans ( 22). Nevertheless, the issue of safety remains to be fully determined. The development of biodegradable supports and the increasing availability of defined MHC class I/class II–restricted tumor-specific peptides ( 41) and HLA-class I/class II recombinant molecules ( 42), together with the possibility of combining class I–restricted and class II–restricted antigenic determinant and different costimulatory ( 43, 44) and inhibitory ( 45) receptors, or immunomodulating agents (i.e., cytokines) might provide the unique possibility of generating “ad-hoc” and “off-the-shelf” reagents suitable for different clinical purposes.

Acknowledgments

Grant support: Associazione Italiana per la Ricerca sul Cancro, Compagnia di San Paolo, Istituto Mobiliare Italiano, Ministero dell'Istruzione, dell'Università e della Ricerca, Fondo per gli Investimenti della Ricerca di Base (RBNE017B4C_006) and the European Community (contract LSHC-CT-2005-018914 “ATTACK”). S. Caserta was supported by a fellowship from the International Ph.D. Program in Molecular Medicine and P. Alessi by a fellowship from the Associazione Italiana per la Ricerca sul Cancro.

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.

We thank Dr. Robin Stephens, Dr. Robert Salmond, and Dr. Valentino Parravicini (National Institute for Medical Research) for critical suggestions and help given during the editing process.

Footnotes

  • Note: Current address for S. Caserta: National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.

  • ↵3 Unpublished results.

  • Received October 8, 2007.
  • Revision received December 27, 2007.
  • Accepted February 7, 2008.
  • ©2008 American Association for Cancer Research.

References

  1. ↵
    Hung K, Hayashi R, Lafond-Walker A, Lowenstein C, Pardoll D, Levitsky H. The central role of CD4(+) T cells in the antitumor immune response. J Exp Med 1998; 188: 2357–68.
    OpenUrlAbstract/FREE Full Text
  2. Marzo AL, Kinnear BF, Lake RA, et al. Tumor-specific CD4(+) T cells have a major “post-licensing” role in CTL mediated anti-tumor immunity. J Immunol 2000; 165: 6047–55.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Benigni F, Zimmermann VS, Hugues S, et al. Phenotype and homing of CD4 tumor-specific T cells is modulated by tumor bulk. J Immunol 2005; 175: 739–48.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Topalian SL, Gonzales MI, Parkhurst M, et al. Melanoma-specific CD4+ T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes. J Exp Med 1996; 183: 1965–71.
    OpenUrlAbstract/FREE Full Text
  5. Manici S, Sturniolo T, Imro MA, et al. Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11 [see comments]. J Exp Med 1999; 189: 871–6.
    OpenUrlAbstract/FREE Full Text
  6. Pieper R, Christian RE, Gonzales MI, et al. Biochemical identification of a mutated human melanoma antigen recognized by CD4(+) T cells [see comments]. J Exp Med 1999; 189: 757–66.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Crosti M, Longhi R, Consogno G, Melloni G, Zannini P, Protti MP. Identification of novel subdominant epitopes on the carcinoembryonic antigen recognized by CD4+ T cells of lung cancer patients. J Immunol 2006; 176: 5093–9.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Staveley-O'Carroll K, Sotomayor E, Montgomery J, et al. Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc Natl Acad Sci U S A 1998; 95: 1178–83.
    OpenUrlAbstract/FREE Full Text
  9. Sotomayor EM, Borrello I, Tubb E, et al. Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat Med 1999; 5: 780–7.
    OpenUrlCrossRefPubMed
  10. ↵
    Zimmermann VS, Casati A, Schiering C, et al. Tumors hamper the immunogenic competence of CD4+ T cell-directed dendritic cell vaccination. J Immunol 2007; 179: 2899–909.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998; 4: 328–32.
    OpenUrlCrossRefPubMed
  12. Schultz ES, Schuler-Thurner B, Stroobant V, et al. Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy. J Immunol 2004; 172: 1304–10.
    OpenUrlAbstract/FREE Full Text
  13. Ossendorp F, Mengede E, Camps M, Filius R, Melief CJ. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J Exp Med 1998; 187: 693–702.
    OpenUrlAbstract/FREE Full Text
  14. Casares N, Lasarte JJ, de Cerio AL, et al. Immunization with a tumor-associated CTL epitope plus a tumor-related or unrelated Th1 helper peptide elicits protective CTL immunity. Eur J Immunol 2001; 31: 1780–9.
    OpenUrlCrossRefPubMed
  15. Miyazawa M, Fujisawa R, Ishihara C, et al. Immunization with a single T helper cell epitope abrogates Friend virus-induced early erythroid proliferation and prevents late leukemia development. J Immunol 1995; 155: 748–58.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Hanson HL, Kang SS, Norian LA, Matsui K, O'Mara LA, Allen PM. CD4-directed peptide vaccination augments an antitumor response, but efficacy is limited by the number of CD8+ T cell precursors. J Immunol 2004; 172: 4215–24.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Banchereau J, Palucka AK, Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 2005; 5: 296–306.
    OpenUrlCrossRefPubMed
  18. ↵
    Suhoski MM, Golovina TN, Aqui NA, et al. Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules. Mol Ther 2007; 15: 981–8.
    OpenUrlCrossRefPubMed
  19. ↵
    Trickett A, Kwan YL. T cell stimulation and expansion using anti-CD3/CD28 beads. J Immunol Methods 2003; 275: 251–5.
    OpenUrlCrossRefPubMed
  20. ↵
    Curtsinger J, Deeths MJ, Pease P, Mescher MF. Artificial cell surface constructs for studying receptor-ligand contributions to lymphocyte activation. J Immunol Methods 1997; 209: 47–57.
    OpenUrlCrossRefPubMed
  21. ↵
    Tham EL, Jensen PL, Mescher MF. Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins. J Immunol Methods 2001; 249: 111–9.
    OpenUrlCrossRefPubMed
  22. ↵
    Mitchell MS, Kan-Mitchell J, Morrow PR, Darrah D, Jones VE, Mescher MF. Phase I trial of large multivalent immunogen derived from melanoma lysates in patients with disseminated melanoma. Clin Cancer Res 2004; 10: 76–83.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Oelke M, Maus MV, Didiano D, June CH, Mackensen A, Schneck JP. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med 2003; 9: 619–24.
    OpenUrlCrossRefPubMed
  24. ↵
    Maus MV, Riley JL, Kwok WW, Nepom GT, June CH. HLA tetramer-based artificial antigen-presenting cells for stimulation of CD4+ T cells. Clin Immunol 2003; 106: 16–22.
    OpenUrlCrossRefPubMed
  25. ↵
    van Rensen AJ, Wauben MH, Grosfeld-Stulemeyer MC, van Eden W, Crommelin DJ. Liposomes with incorporated MHC class II/peptide complexes as antigen presenting vesicles for specific T cell activation. Pharm Res 1999; 16: 198–204.
    OpenUrlCrossRefPubMed
  26. ↵
    Prakken B, Wauben M, Genini D, et al. Artificial antigen-presenting cells as a tool to exploit the immune ‘synapse’. Nat Med 2000; 6: 1406–10.
    OpenUrlCrossRefPubMed
  27. ↵
    Rogers J, Mescher MF. Augmentation of in vivo cytotoxic T lymphocyte activity and reduction of tumor growth by large multivalent immunogen. J Immunol 1992; 149: 269–76.
    OpenUrlAbstract
  28. Mescher MF, Savelieva E. Stimulation of tumor-specific immunity using tumor cell plasma membrane antigen. Methods 1997; 12: 155–64.
    OpenUrlCrossRefPubMed
  29. ↵
    Goldberg J, Shrikant P, Mescher MF. In vivo augmentation of tumor-specific CTL responses by class I/peptide antigen complexes on microspheres (large multivalent immunogen). J Immunol 2003; 170: 228–35.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Maus MV, Kovacs B, Kwok WW, et al. Extensive replicative capacity of human central memory T cells. J Immunol 2004; 172: 6675–83.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Malherbe L, Filippi C, Julia V, et al. Selective activation and expansion of high-affinity CD4+ T cells in resistant mice upon infection with Leishmania major. Immunity 2000; 13: 771–82.
    OpenUrlCrossRefPubMed
  32. ↵
    Murphy KM, Heimberger AB, Loh DY. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 1990; 250: 1720–3.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Camporeale A, Boni A, Iezzi G, et al. Critical impact of the kinetics of dendritic cells activation on the in vivo induction of tumor-specific T lymphocytes. Cancer Res 2003; 63: 3688–94.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Lucas B, Vasseur F, Penit C. Normal sequence of phenotypic transitions in one cohort of 5-bromo-2′-deoxyuridine-pulse-labeled thymocytes. Correlation with T cell receptor expression. J Immunol 1993; 151: 4574–82.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods 1994; 171: 131–7.
    OpenUrlCrossRefPubMed
  36. ↵
    Bradley LM, Haynes L, Swain SL. IL-7: maintaining T-cell memory and achieving homeostasis. Trends Immunol 2005; 26: 172–6.
    OpenUrlCrossRefPubMed
  37. ↵
    Klebanoff CA, Gattinoni L, Torabi-Parizi P, et al. Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci U S A 2005; 102: 9571–6.
    OpenUrlAbstract/FREE Full Text
  38. Gattinoni L, Klebanoff CA, Palmer DC, et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8(+) T cells. J Clin Invest 2005; 115: 1616–26.
    OpenUrlCrossRefPubMed
  39. ↵
    Harari A, Vallelian F, Meylan PR, Pantaleo G. Functional heterogeneity of memory CD4 T-cell responses in different conditions of antigen exposure and persistence. J Immunol 2005; 174: 1037–45.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Zimmermann VS, Benigni F, Mondino A. Immune surveillance and anti-tumor immune responses: an anatomical perspective. Immunol Lett 2005; 98: 1–8.
    OpenUrlCrossRefPubMed
  41. ↵
    Kessler JH, Melief CJ. Identification of T-cell epitopes for cancer immunotherapy. Leukemia 2007; 21: 1859–74.
    OpenUrlCrossRefPubMed
  42. ↵
    Mallone R, Nepom GT. MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete. Clin Immunol 2004; 110: 232–42.
    OpenUrlCrossRefPubMed
  43. ↵
    Rudolf D, Silberzahn T, Walter S, et al. Potent costimulation of human CD8 T cells by anti-4–1BB and anti-CD28 on synthetic artificial antigen presenting cells. Cancer Immunol Immunother 2007; 53: 175–83.
    OpenUrl
  44. ↵
    Zhang H, Snyder KM, Suhoski MM, et al. 4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy. J Immunol 2007; 179: 4910–8.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Griffin MD, Hong DK, Holman PO, et al. Blockade of T-cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152). J Immunol 2000; 164: 4433–42.
    OpenUrlAbstract/FREE Full Text
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April 2008
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Synthetic CD4+ T Cell–Targeted Antigen-Presenting Cells Elicit Protective Antitumor Responses
Stefano Caserta, Patrizia Alessi, Jlenia Guarnerio, Veronica Basso and Anna Mondino
Cancer Res April 15 2008 (68) (8) 3010-3018; DOI: 10.1158/0008-5472.CAN-07-5796

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Synthetic CD4+ T Cell–Targeted Antigen-Presenting Cells Elicit Protective Antitumor Responses
Stefano Caserta, Patrizia Alessi, Jlenia Guarnerio, Veronica Basso and Anna Mondino
Cancer Res April 15 2008 (68) (8) 3010-3018; DOI: 10.1158/0008-5472.CAN-07-5796
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