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The Journal of Immunology, 2007, 179, 2899 -2909
Copyright © 2007 by The American Association of Immunologists, Inc.
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Tumors Hamper the Immunogenic Competence of CD4+ T Cell-Directed Dendritic Cell Vaccination1

Valérie S. Zimmermann2,*, Anna Casati3,*, Chris Schiering*,{dagger}, Stefano Caserta*,{ddagger}, Rodrigo Hess Michelini*,§, Veronica Basso* and Anna Mondino4,*

* Cancer Immunotherapy and Gene Therapy Program, San Raffaele Scientific Institute, Department of Biotechnology, Milan, Italy; {dagger} University of Glasgow, Scotland, United Kingdom; {ddagger} University "Vita e Salute", Milan, Italy; and § Open University and University "Vita e Salute", Milan, Italy.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells loaded with tumor-derived peptides induce protective CTL responses and are under evaluation in clinical trails. We report in this study that prophylactic administration of dendritic cells loaded with a MHC class II-restricted peptide derived from a model tumor Ag (Leishmania receptor for activated C kinase (LACK)) confers protection against LACK-expressing TS/A tumors, whereas therapeutic vaccination fails to cure tumor-bearing mice. Although CD4+ T cell-directed dendritic cell vaccination primed effector-like (CD44highCD62Llow, IL-2+, IFN-{gamma}+) and central memory-like lymphocytes (CD44highCD62Lhigh, only IL-2+) in tumor-free mice, this was not the case in tumor-bearing animals in which both priming and persistence of CD4+ T cell memory were suppressed. Suppression was specific for the tumor-associated Ag LACK, and did not depend on CD25+ T cells. Because T cell help is needed for protective immunity, we speculate that the ability of tumors to limit vaccine-induced CD4+ T cell memory could provide a partial explanation for the limited efficacy of current strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The identification of genes encoding for tumor-specific Ag and the isolation of tumor-specific lymphocytes in cancer patients (1, 2) have encouraged the development of several immunotherapeutic strategies aimed at boosting natural antitumor immune responses (3, 4). Trials in cancer patients have demonstrated that vaccine-specific CD8+ T cells capable of IFN-{gamma} production accumulate in patients in response to vaccination (5, 6). However, responses are often short-lived (7) and do not always correlate with objective tumor regression (8). Although most of the vaccination studies conducted to date have focused on CD8-directed immunization (8, 9), it is now clear that optimal induction and maintenance of CD8 cytotoxic effector T lymphocytes require CD4+ T cell help. Th cells favor APC maturation and Ag cross-priming (10, 11, 12), and the generation of CD8+ memory T cells (13, 14, 15, 16, 17). Furthermore, CD4+ T cells mediate the recruitment and the activation of macrophages and eosinophils at the tumor site (18, 19), and exert antiangiogenic activity through secretion of soluble factors (such as IFN-{gamma}) (20). Accordingly, CD4-directed vaccination in some instances was reported to augment antitumor responses (21, 22, 23, 24).

We have recently characterized a natural antitumor CD4+ T cell response by tracing T cells specific for the model Ag Leishmania receptor for activated C kinase (LACK)5 in mice bearing LACK-expressing tumors. Although the tumor efficiently induced the differentiation of IFN-{gamma}-producing effector CD4 T cells capable of seeding the nonlymphoid organs and the tumor, it hampered the accumulation of central memory T lymphocytes able to persist in the lymphoid compartment (25). In contrast to tumor cells, dendritic cells (DC) loaded with a LACK derived peptide-primed systemic CD4+ central memory T cell responses in tumor-free hosts (25, 26). Because DC vaccination is considered one of the most promising strategies in clinical trials (27, 28, 29), in this study we have compared the potency of DC-based vaccination in prophylactic and therapeutic tumor settings. The results indicate that immunization of tumor-free mice with peptide-loaded DC elicits tumor-specific effector and central memory CD4+ T lymphocytes and confers protection against subsequent tumor challenge. In contrast, vaccination of tumor-bearing mice induces limited numbers of tumor-specific effector T cells, and fails to generate central memory lymphocytes and to cure the mice. Tumor-mediated immune suppression is Ag specific, does not involve CD25+ cells, and parallels the accumulation of CD11b+Gr1+ myeloid suppressor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cells

Eight- to 10-wk-old BALB/c mice were purchased from Charles River Laboratories. The 16.2β and DO11.10 transgenic (Tg) BALB/c mice were previously described (30, 31). Briefly, T lymphocytes derived from 16.2β mice express a Tg TCR β-chain-specific I-Ad-restricted peptide derived from the Leishmania major-derived Ag LACK (FSPSLEHPIVVSGSWD), whereas those derived from DO11.10 mice express a Tg TCR ({alpha}β) specific for an I-Ad-restricted peptide derived from chicken OVA (ISQAVHAAHAEINEAGR). Where indicated, 3 x 106 DO11.10 T cells were adoptively transferred by i.v. injection into syngeneic 16.2β mice and analyzed, as previously described (32).

TS/A mouse mammary adenocarcinoma cells (33) were grown in vitro at 37°C in complete medium (RPMI 1640-10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2.5 x 10–5 M 2-ME; Invitrogen Life Technologies). TS/A-LACK cells were obtained by stable transfection of TS/A cells with a pcDNA3-derived vector encoding for a truncated form of the L. major-derived model Ag LACK, and limiting dilution of G418-resistant clones (25). In these cells, LACK is expressed as a cytosolic protein and is representative of a tumor-associated Ag. Bone marrow-derived DC were obtained, as previously described (34). Briefly, bone marrow precursors were propagated for 7 days in complete Iscove’s medium containing 25 ng/ml mouse rGM-CSF and 5 ng/ml mouse rIL-4 (Bender MedSystems). Bone marrow-derived DC 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 µM class II-restricted immunodominant peptide LACK (FSPSLEHPIVVSGSWD). 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).

Immunization

BALB/c mice were vaccinated s.c. in the left flank with 2 x 105 matured bone marrow-derived DC loaded with the LACK immunodominant CD4 peptide (FSPSLEHPIVVSGSWD) (DC-LACK) and boosted 10 days later with DC-LACK in the opposite flank. Vaccinated mice were then challenged s.c. into the right flank with 4 x 105 exponentially growing TS/A or TS/A-LACK cells suspended in 100 µl of PBS and injected. Tumor growth was monitored by measuring tumor diameters every other day using a metric caliper (25). Animals were scored as tumor positive if the mean of the three diameters of the tumor was >2 mm, and were sacrificed when the tumor reached a diameter >10 mm or ulcerated.

To evaluate the effect of vaccination on established TS/A-LACK tumors, 4 x 105 exponentially growing TS/A-LACK tumor cells were injected in the left flank. Tumors were allowed to grow for 5 days. Thereafter, mice were immunized in the opposite flank with 2 x 105 DC-LACK cells, and tumor growth was evaluated, as described above.

All of the in vivo studies were approved by the Ethical Committee of the San Raffaele Scientific Institute and performed according to its guideline.

Flow cytometry analysis

Mice were sacrificed, and the draining, nondraining lymph nodes (LN) (axillary, brachial, and inguinal), and the spleen were collected by surgical resection. LN and spleen were homogenized into a single-cell suspension. I-Ad/LACK multimer staining was performed, as previously described. Briefly, 6 x 105 cells were first incubated with a blocking buffer (5% rat serum + 95% culture supernatant of 2.4G2 anti-FcR mAb-producing hybridoma cells, 20 min) and then stained with the I-Ad/LACK multimers (3 µg/sample, 1 h at 4°C, in PBS supplemented with 0.5% BSA). The cells were then stained with anti-CD4, anti-CD44, anti-CD11b, anti-B220, anti-CD8a mAbs (BD Pharmingen), and TO-PRO-3 (1 nM; Molecular Probes). CD8a+, CD11b+, B220+, TOPRO+ cells were excluded by electronic gating during the acquisition. One thousand CD4+, I-Ad/LACK+ T cells were generally collected (FACSCalibur; BD Biosciences) and analyzed using the CellQuest Software. Where indicated, LN and spleen cells were stained with anti-Ly6G, anti-Ly6C (Gr1), and anti-CD11b mAb, or with anti-CD4 and anti-CD25 mAb (BD Biosciences). In some instances, intracellular FoxP3 expression was evaluated according to manufacturer’s instruction (eBioscience).

Intracellular cytokine staining

Intracellular cytokine content was determined, as previously described (35). Briefly, 1 x 106 LN cells from vaccinated and tumor-free mice were incubated with 5 x 106 DO11.10 splenocytes pulsed or not with 2 µM LACK peptide. After 2 h in culture at 37°C, brefeldin A (10 µg/ml; Sigma-Aldrich) was added. Following an additional 2 h, control and restimulated cells were surface stained with anti-CD4 and KJ1-26 (the anti-clonotypic mAb recognizing the Tg TCR expressed by DO11.10 mice used to exclude spleen-derived CD4+ T cells) 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 and anti-IFN-{gamma} mAb in permeabilization buffer. Fifty thousand CD4+, KJ1-26– events were generally collected (FACSCalibur; BD Biosciences) and analyzed using the CellQuest software.

Statistical analyses

Statistical analyses were performed using either two-tailed Student’s t test or {chi}2. Results were considered statistically significant when p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Vaccination with DC loaded with a tumor-specific MHC class II-restricted peptide protects mice from tumor growth, but fails to cure established tumors

TS/A adenocarcinoma cells expressing a cytosolic fragment of the L. major-derived Ag LACK elicit the development of solid tumors in syngeneic mice (25). In a previous study, we found that tumor (LACK)-specific CD4+ T cell responses are spontaneously generated in the presence of TS/A-LACK tumors, and contribute to the control of tumor growth, but are insufficient to elicit tumor rejection. This correlated with the absence of tumor-specific CD4+ central memory-like T lymphocytes (25). In contrast to tumors, bone marrow-derived DC, matured by LPS activation (34) and pulsed with the class II-restricted LACK immunodominant peptide, elicit systemic CD4+ T cell memory (25). We thus sought to investigate whether this strategy could confer antitumor protection. Unpulsed and LACK-pulsed DC (2 x 105) were injected s.c. in BALB/c mice (Fig. 1). Mice were boosted after 10 days, and after an additional 7 days were challenged with 4 x 105 viable TS/A-LACK tumor cells (a number representing >20 times the minimal tumorigenic dose; our unpublished data). Although TS/A-LACK tumors rapidly developed in control mice, tumor growth was not observed in 80% of DC-LACK-vaccinated mice (Fig. 1, A and B). The remaining 20% of the mice developed tumors as control mice (Fig. 1A). Tumor protection was statistically significant (p < 0.001), and was lost upon depletion of CD4+ T cells (data not shown). Protected mice were rechallenged with TS/A-LACK tumor cells in the opposite flank. In all cases, the mice rejected the second tumor challenge (data not shown), suggesting that the rejection of the primary tumor conferred protection against the secondary tumor challenge. In an independent experiment, mice were vaccinated and challenged with TS/A-LACK tumor cells 60 days later, an interval sufficient for memory establishment. Although TS/A-LACK tumors rapidly developed in all of the mice vaccinated with control DC, DC-LACK-vaccinated mice showed delayed tumor appearance (Fig. 1C). Moreover, 60% of these mice remained tumor free for up to 80 days (Fig. 1D). These data indicate that vaccination with DC pulsed with a class II-restricted tumor-specific peptide elicits long-lasting protective immunity endowed with memory.


Figure 1
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  		FIGURE 1. Effect of DC vaccination on tumor growth in BALB/c mice: protection and cure. A–D, Mice were vaccinated twice with 2 x 105 unpulsed (empty symbols) or LACK-pulsed DC (filled symbols) and challenged with 4 x 105 viable TS/A-LACK 10 days (A and B) and 60 days (C and D) after vaccination. E and F, Mice were challenged with 4 x 105 TS/A-LACK in the flank, and 5 days later, vaccinated ({blacktriangleup}) or not ({square}) with LACK-pulsed DC in the opposite flank. Tumor growth was monitored at the indicated times by measuring the tumor diameters using a metric caliper, and is reported as mean tumor volume ± SD (A, C, and E). Mice were sacrificed when the tumor reached a diameter >10 mm or ulcerated, and the percentage of living mice is indicated (B, D, and F). A, LACK-vaccinated mice were divided in protected (•) and not protected mice ({blacktriangleup}). Statistical significance is as follows: ***, p < 0.001.

 
We then investigated whether DC-LACK vaccination could be used to cure tumor-bearing mice. To this aim, 4 x 105 TS/A-LACK tumor cells were injected in BALB/c mice. Five days later, a time required for a detectable tumor to develop, 2 x 105 control DC or DC-LACK were injected in the contralateral flank. In contrast to preventive vaccination, therapeutic vaccination of tumor-bearing mice failed to induce an immune response able to control tumor growth. Indeed, TS/A-LACK tumors developed with comparable kinetics in both control and vaccinated mice (Fig. 1E) and caused death of the mice at similar times (Fig. 1F).

DC-LACK vaccination primes effector and central memory LACK-specific CD4+ T cells

In the attempt to identify the reasons for the differences in clinical outcome, we compared vaccine-induced CD4+ T cell responses in 16.2β Tg mice both in prophylactic and therapeutic settings. The 16.2β Tg mice express a LACK-specific Tg TCR β-chain, which in combination with endogenously rearranged TCR {alpha}-chains generate a polyclonal population of CD4+ LACK-specific T cells (0.5–1% of CD4+ T cells) detectable by fluorescent I-Ad/LACK multimers (30). This model has allowed to enumerate naive and memory LACK-specific CD4+ T cells during L. major infection (30), and TS/A-LACK tumor development (25). We first analyzed T cell responses to DC-LACK vaccination in naive mice. DC pulsed with the LACK peptide or with an irrelevant class II-restricted OVA peptide (DC-OVA) were s.c. injected into 16.2β mice, which were then sacrificed 6 and 15 days later. LN draining and distal (nondraining) to the site of vaccination were surgically excised, and cells were analyzed by flow cytometry after staining with anti-CD4 mAb and the I-Ad/LACK multimer. Vaccination with DC-LACK resulted in the rapid and selective accumulation of I-Ad/LACK+ CD4+ T cells in the draining LN. By day 6, the frequency (2.23% ± 0.91) and total number (99.9 ± 50.2 x 103) of I-Ad/LACK+ CD4+ T cells in the draining LN of DC-LACK-vaccinated mice were significantly higher than the ones found in control mice (0.56% ± 0.09 and 12.4 ± 1.9 x 103; p < 0.001 and p = 0.015, respectively; Fig. 2, A and B), where they remained comparable to those of naive mice (data not shown). By day 15, the frequency and total number of LACK-specific T cells decreased (0.64% ± 0.13, and 20.6 ± 6.3 x 103), but remained higher than the ones found in control mice (Fig. 2, A and B; p < 0.001 and p = 0.006, respectively). At difference with the draining LN, neither the frequency nor the total number of I-Ad/LACK+ CD4+ T cells found in the LN distal to the site of DC-LACK vaccination (nondraining LN) was statistically different from the ones found in control mice (Fig. 2, C and D).


Figure 2
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  		FIGURE 2. Frequency and total number of LACK-specific CD4+ T cells in LACK-DC-vaccinated 16.2β Tg mice. The 16.2β Tg mice were vaccinated with 2 x 105 OVA-pulsed (DC-OVA, control) or LACK-pulsed DC (DC-LACK). At the indicated times, cells were isolated from axillary, brachial, and inguinal LN draining (dLN; A and B) and nondraining (ndLN; C and D) the site of vaccination. LN cells were stained with I-Ad/LACK multimers and anti-CD4 mAb. The percentage (A and C) and total numbers (B and D) of I-Ad/LACK+ T cells were measured by flow cytometry after gating on viable CD4+, B220–, CD8–, CD11b–, TOPRO-3– cells (see Materials and Methods). Data represent the arithmetic mean ± SD of at least three mice. Statistical significance is as follows: ***, p < 0.001; **, p < 0.01. Experiments were repeated at least four times with similar results.

 
At day 6, a sizeable fraction of I-Ad/LACK+ T cells in the LN draining the site of DC-LACK vaccination expressed high levels of CD69 (31.3% ± 14.1) (Fig. 3A) and CD44 (53.9% ± 17.2) (Fig. 3B). Among the I-Ad/LACK+ T cells, 24% of the cells were CD62Lhigh, whereas 57% (up to 70% of CD44high cells) had down-regulated the surface expression of this LN-homing molecule (Fig. 3C). At day 15, a considerable portion of I-Ad/LACK+ T cells found in the LN draining the site of DC-LACK injection still expressed an activated phenotype (21% ± 5.2 of CD69+ and 41% ± 8.9 CD44high) (Fig. 3, A and B), and CD62Lhigh and CD62Llow were equally distributed among CD44high memory T cells (Fig. 3C). I-Ad/LACK+ CD4+ T cells found in the LN of control DC-OVA-vaccinated mice preserved a naive phenotype because they expressed low levels of CD69 (Fig. 3A) and CD44 (Fig. 3B) and high levels of CD62L (Fig. 3C). Although the frequency and total number of I-Ad/LACK+ T cells remained unchanged, the nondraining LN were enriched of I-Ad/LACK+ CD44high memory T cells, most of which were CD62Lhigh (Fig. 3C). By day 15, LACK-specific CD44high, CD62Lhigh memory cells appeared to be equally distributed between the draining and nondraining LN of vaccinated mice, as revealed by their comparable frequency (22.9% ± 3.3 and 20.1% ± 5.9, correlation = 0.8), which was higher than the one found in control mice (15.0 ± 0.2%).


Figure 3
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  		FIGURE 3. Phenotype of LACK-specific CD4+ T cells in DC-LACK-vaccinated mice. The 16.2β Tg mice were vaccinated with 2 x 105 OVA (DC-OVA control)- or LACK-pulsed DC (DC-LACK). Cells from vaccine-draining LN (dLN) and from nondraining LN (ndLN) were isolated 6 or 15 days after vaccination and stained with I-Ad/LACK multimers, anti-CD4 mAb, and either anti-CD69 or anti-CD44 and anti-CD62L mAb. A and B, Representative flow cytometry profiles are shown after gating on CD4+, B220–, CD8–, CD11b–, TOPRO-3– cells. The frequencies of I-Ad/LACK+ CD4+ cells are indicated in bold. The proportions of CD69+ (A) or CD44high (B) among I-Ad/LACK+ CD4+ cells are indicated in parentheses. C, Representative flow cytometry profiles are depicted after gating on I-Ad/LACK+ CD4+ T cells. Relative frequencies are indicated. Experiments were repeated at least four times with similar results.

 
Next, cytokine secretion by intracellular cytokine staining was analyzed (Fig. 4). Upon LACK restimulation, LN cells derived from DC-LACK-vaccinated mice, and not from those derived from control mice, produced significant levels of IL-2 and/or IFN-{gamma} (dLN; Fig. 4A). By day 6, both IL-2- and IFN-{gamma}-producing cells were detected in the LN draining the site of DC-LACK vaccination. By day 15, the frequency of cytokine-producing cells had decreased, and the draining LN mainly contained IL-2-producing cells (dLN; Fig. 4B). Moreover, the LN distal to the site of DC vaccination revealed a sizeable population of LACK-specific T cells at both days 6 and 15, which was mainly comprised of IL-2-producing cells (ndLN; Fig. 4, A and B). The percentage of cytokine-producing cells highly correlated with the frequency of CD44high I-Ad/LACK+ T cells (Fig. 4, C and D, correlation = 0.9). IL-4, IL-5, and IL-10 were below the limit of detection in all of the experiments (data not shown).


Figure 4
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  		FIGURE 4. Cytokine secretion upon LACK restimulation in DC-LACK-vaccinated mice. The 16.2β Tg mice were vaccinated with DC-OVA or DC-LACK as in Fig. 3 and sacrificed 6 (A and C) or 15 days (B and D) later. Cells derived from the draining (dLN) and nondraining (ndLN) LN were incubated for 4 h with DO11.10 splenocytes in the absence (APC) or in the presence of LACK peptide (APC/LACK). Brefeldin A was added in the last 2 h of incubation. Cells were then stained with anti-CD4 and with the KJ1-26 mAb, fixed, permeabilized, and stained with anti-IL-2 and anti-IFN-{gamma} mAb. A and B, Representative dot plots are shown after gating on CD4+ KJ1-26– cells (to exclude T cells of DO11.10 spleen origin). The frequencies of cells within each quadrant are indicated. C and D, The mean percentage of cytokine (IL-2 and IFN-{gamma})-producing cells detected in unpulsed (APC; {square}) or LACK-pulsed cultures (APC/LACK; {blacksquare}) is compared with the mean percentage of I-Ad/LACK+ CD44highCD4+ T cells (Figure 4) obtained by the flow cytometry analysis depicted in Fig. 3. Data represent the mean of at least four mice and are representative of at least three independent experiments. Statistical significance is as follows: **, p < 0.01; *, p < 0.05.

 
Together these data indicate that DC-LACK vaccination primes LACK-specific memory cells with a phenotype consistent with effector-like (IFN-{gamma}-producing, CD44high, CD62Llow) and central memory-like (IL-2-producing CD44high, CD62Lhigh) (36, 37, 38) lymphocytes, a fraction of which is able to recirculate within the lymphoid system.

Established LACK-expressing tumors hamper the accumulation of DC-induced LACK-specific CD4+ T cell memory

To understand the reasons for the failure of DC-LACK vaccines to confer protection in therapeutic settings, we next visualized the fate of LACK-specific CD4+ T cell vaccination in tumor-bearing mice. To this aim, 4 x 105 TS/A-LACK tumor cells were injected in the right flank of 16.2β mice. Five days later, tumor-bearing and naive control mice received a s.c. injection of 2 x 105 DC-LACK in the flank contralateral to the site of tumor challenge. Six and 15 days after vaccination, cells were recovered from the LN draining the site of vaccination and analyzed by flow cytometry following staining with the I-Ad/LACK multimer, anti-CD4 mAb, and anti-CD44 mAb. Results show that whereas vaccination of naive mice elicited the expected expansion of LACK-specific T cells, lower frequencies (data not shown) and total numbers of I-Ad/LACK+ CD4+ T cells (Fig. 5A) and of I-Ad/LACK+ CD4+CD44high memory T cells (Fig. 5B) were observed in vaccinated TS/A-LACK tumor-bearing mice. This was evident at both days 6 and 15 after vaccination. In contrast to TS/A-LACK tumor-bearing animals, TS/A tumor-bearing mice responded to DC-LACK vaccination to extents comparable of control-vaccinated mice, both at day 6 (Fig. 8) and day 15 (Figs. 5, 6, and 7). Tumor-reactive LN were also analyzed (Fig. 5, C and D). Comparable numbers of I-Ad/LACK+ CD4+ T cells (Fig. 5C) and of I-Ad/LACK+ CD4+CD44high memory T cells (Fig. 5D) were found in the tumor-draining LN of TS/A-LACK tumor-bearing mice either untreated or vaccinated with DC-LACK. Thus, reduced DC-LACK-mediated CD4+ T cell priming in TS/A-LACK tumor-bearing mice appears to be due to the inhibition of T cell priming, rather than to the sequestration of primed T cells in tumor-reactive LN.


Figure 5
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  		FIGURE 5. Effects of tumor growth on DC-LACK CD4+ T cell priming. The 16.2β Tg mice were challenged in the right flank with 4 x 105 TS/A or TS/A-LACK tumor cells or left untreated (Naive). Five days later, tumor-free and tumor-bearing mice were left untreated or vaccinated in the flank opposite to the tumor with 2 x 105 LACK-pulsed DC. Six and 15 days later, mice were sacrificed and cells were isolated from the axillary, brachial, and inguinal LN draining the site of vaccination (Vaccine-dLN; A and B) or from the tumor-draining LN (Tumor-dLN; C and D) and were stained with I-Ad/LACK multimers, anti-CD4, anti-CD44, and anti-CD62L mAb, and analyzed by flow cytometry. The mean total number ± SD of I-Ad/LACK+ CD4+ T cells (A and C) and of I-Ad/LACK+ CD44highCD4+ T cells (B and D) is shown. Data represent the mean of at least four mice and are representative of at least five independent experiments.

 

Figure 8
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  		FIGURE 8. Immunosuppression is restricted to tumor-associated Ags, and is not broken by increasing the potency of DC vaccination. A–C, Tumor-free 16.2β mice and 16.2β mice bearing 5-day-old TS/A or TS/A-LACK tumors were i.v. adoptively transferred with 3 x 106 DO11.10 TCR Tg T cells. Seeding of the cells (AT only) was comparable among the groups. A day later, mice were vaccinated with a mixture of 2 x 105 LACK-pulsed DC and 2 x 105 OVA-pulsed DC. After 5 days, mice were sacrificed and the LN draining the site of vaccination were analyzed for the presence of LACK-specific and OVA-specific memory cells by flow cytometry following staining with anti-CD4, anti-CD44, and I-Ad/LACK multimers or anti-CD4 and KJ1-26 anti-clonotypic mAb, respectively. A, Schematic representation of the experiment. B and C, The total numbers ± SD of I-Ad/LACK+ CD4+CD44high T cells (B) or CD4+ KJ1-26+ T cells (C) are depicted. Data represent the mean of three mice per group and are representative of two independent experiments. D, Tumor-free and TS/A-LACK tumor-bearing mice were vaccinated with 2 x 105 or 106 LACK-pulsed DC. The mean ± SD of total numbers of I-Ad/LACK+ CD4+CD44high T cells at day 5 obtained by flow cytometry is depicted.

 

Figure 6
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  		FIGURE 6. LACK-specific CD4+ T cells found in TS/A tumor-bearing mice remain responsive to LACK. The 16.2β Tg mice were challenged in the right flank with 4 x 105 TS/A or TS/A-LACK tumor cells or left untreated (Tumor-free (Naive)). Five days later, cells were isolated from axillary, brachial, and inguinal LN draining (dr LN) or nondraining (ndr LN) the site of tumor growth and stained with I-Ad/LACK multimers, anti-CD4, anti-CD44, and anti-CD62L mAb. A, Events are shown after gating of viable CD4+ T cells. The frequency of I-Ad/LACK+ T cells among viable CD4+ cells is indicated. B and C, Events reflect viable CD4+ I-Ad/LACK+. D, Cells derived from naive LN and from tumor-nondraining LN were stimulated in vitro for 48 h with irradiated syngenic BALB/c splenocytes and the indicated amount of LACK peptide. Proliferation was then measured in triplicate cultures by a 20-h pulse with [3H]thymidine. E, IL-2 and IFN-{gamma} release was measured in culture supernatants by capture ELISA after a 24 and 48h of culture, respectively.

 

Figure 7
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  		FIGURE 7. TS/A-LACK tumors prevent the development and recirculation of LACK-specific memory CD4 T cells. LN distal to the site of DC injection and of tumor growth (popliteal LN, tumor/vaccine distal LN) were derived from vaccinated tumor-free animals, from vaccinated tumor-bearing mice, and from unvaccinated TS/A-LACK tumor-bearing mice, and analyzed by flow cytometry 15 days after vaccination. Cells were analyzed following staining with I-Ad/LACK multimers, anti-CD4, anti-CD44, and anti-CD62L mAb (A and C) and by intracellular cytokine staining after LACK restimulation, as described in Fig. 4 (B and D). A and B, Representative dot plots are shown after gating on CD4+, B220–, CD8–, CD11b– (A), and CD4+ KJ1-26– viable cells (B). The frequency of cells within each quadrant is indicated. C and D, The mean percentage ± SD of CD62Lhigh and CD62Llow cells among I-Ad/LACK+ CD4+CD44high T cells (C) and of LACK-specific IL-2+ or IL-2+/IFN-{gamma}+ CD4+ T cells (D) is depicted. Data represent the mean of at least four mice and are representative of at least three independent experiments.

 
Defective LACK-specific CD4+ T cell responses were not accountable for by T cell unresponsiveness. Indeed, in a previous study, we showed that growing TS/A-LACK tumors support activation of effector memory-like LACK-specific CD4+ T cells rather than inducing T cell anergy. Primed cells are found in tumor-draining LN, blood, and spleen, and within the tumor, but fail to accumulate in tumor-distal LN (25). Accordingly, most of I-Ad/LACK+ CD4+ T cells within tumor-distal LN expressed low levels of CD44 (Fig. 6A) and high levels of CD62L (Fig. 6B), and were small in size (Fig. 6C), as those of naive tumor-free mice, at difference with LACK-specific CD4+ T cells found in TS/A-LACK tumor-draining LN, which expressed high levels of CD44 and low levels of CD62L, and were enlarged in size (Fig. 6) (25). LACK-specific T cells within tumor-nondraining LN also remained responsive to LACK, because they proliferated to levels comparable to those of naive T cells (Fig. 6D) (25), and secreted IL-2 and IFN-{gamma}, to levels that exceeded those produced by naive cells (Fig. 6E).

Growing TS/A-LACK tumors not only hampered DC-mediated T cell priming, but also influenced the persistence and redistribution of cells primed by DC-LACK vaccination (Fig. 7). Although a sizeable population of I-Ad/LACK+ CD44high, CD62Lhigh memory T cells (Fig. 7, A and C) capable of LACK-specific IL-2 secretion (Fig. 7, B and D) was evident 15 days after vaccination in LN distal to the site of vaccination and to the site of tumor growth of vaccinated control mice (DC/LACK) and vaccinated TS/A tumor-bearing (TS/A+DC/LACK) mice, this was not the case in vaccinated TS/A-LACK tumor-bearing mice. In these mice, I-Ad/LACK+ CD4+ T cells revealed a naive phenotype (CD44low, CD62Lhigh) as in the case of TS/A-LACK-unvaccinated control mice (Fig. 7).

Together these data indicate that the presence of LACK-expressing TS/A tumors limits the ability of LACK-pulsed DC to induce LACK-specific T cell memory.

Suppression of DC-induced T cell responses is Ag specific, and cannot be overcome by increasing DC numbers

Profound immunosuppression has been observed in mouse models of tumor disease (39). To verify whether this was the case in TS/A-LACK tumor-bearing mice, naive and tumor-bearing 16.2β mice received an adoptive transfer of OVA-specific DO11.10 TCR Tg CD4+ T cells. Mice were then vaccinated with a mixture of DC pulsed with either LACK or OVA. LACK- and OVA-specific T cell responses were independently enumerated by I-Ad/LACK and anti-clonotypic KJ1-26 Ab staining in the DC-draining LN 5 days after vaccination (a schematic representation of the experiment is reported in Fig. 8A). Although vaccination induced a LACK-specific response in control and TS/A tumor-bearing mice and not in TS/A-LACK tumor-bearing mice (Fig. 8B), it primed OVA-specific KJ1-26+ T cells to a comparable extent in control, TS/A, and TS/A-LACK tumor-bearing mice (Fig. 8C). This is indicative of mechanisms mediating LACK-specific rather than general immunosuppression in TS/A-LACK tumor-bearing mice.

LACK-specific inhibition was observed even upon increasing the potency of vaccination. Both the frequency and total number of LACK-specific CD4+ T cells were markedly increased in tumor-free mice that received 106 DC-LACK as compared with mice that received the lower number of DC (2 x 105) (Fig. 8D). In contrast, vaccination of TS/A-LACK-tumor bearing mice with 1 x 106 DC-LACK mice did not improve LACK-specific responses, and was significantly less effective than vaccination of tumor-free animals with 0.2 x 106 DC-LACK (p = 0.006). Thus, increasing the number of LACK-presenting DC does not overcome the suppressive mechanisms in TS/A-LACK-tumor bearing animals.

The increased frequency of Gr-1+ CD11b+ myeloid cells correlates with tumor-mediated suppression

Several inhibitory mechanisms have been reported to develop in the context of tumor development. Among these, CD4+CD25+ Foxp3+ T cells have been recently associated with tumor-related immunosuppression (40, 41). We thus analyzed the relative representation of CD4+CD25+ Foxp3+ in tumor-free and tumor-bearing mice by flow cytometry. The frequency and total number of CD4+CD25+ Foxp3+ T cells were comparable in the draining, nondraining LN (Fig. 9A) and the spleen (Fig. 9B) of tumor-free and TS/A-LACK tumor-bearing mice. To further investigate the role of these cells in tumor-mediated suppression, tumor-free and TS/A-LACK tumor-bearing mice received i.v. injections of anti-CD25 mAb, reported to deplete circulating CD25+ T cells and in some cases to ameliorate vaccine-induced T cell responses (42, 43). We found that DC-induced LACK-specific CD4+ T cell responses were slightly higher in tumor-free mice depleted of CD25+ cells. However, despite complete depletion of circulating CD25+ cells, DC-LACK failed to induce optimal T cell priming in LN of TS/A-LACK tumor-bearing mice (Fig. 9C).


Figure 9
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  		FIGURE 9. Mechanisms of suppression in TS/A-LACK tumor-bearing mice. A and B, Cells derived from the LN (A) and the spleen (B) of tumor-free and TS/A-LACK tumor-bearing (tumor-draining and tumor-nondraining) 16.2β Tg mice, and analyzed by flow cytometry after staining with anti-CD4, anti-CD25, and anti-FoxP3. The frequency of CD4+CD25+ FoxP3+ T cells is depicted. C, Tumor-free and TS/A-LACK tumor-bearing mice received anti-CD25 mAb 4 and 2 days before vaccination, and at the time of DC-LACK injection. Depletion of the cells was monitored in peripheral blood and found to be >95%. Mice were sacrificed 5 days after vaccination, and LACK-specific T cells were enumerated in vaccine-draining LN. The frequency of CD44high cells among I-Ad/LACK+ CD4+ T cells is depicted. Data represent the mean of three mice per group and are representative of two independent experiments. D, Cells derived from the spleen of tumor-free and TS/A-LACK tumor-bearing 16.2β mice were analyzed by flow cytometry after staining with anti-Gr1 and anti-CD11b cells. Total numbers are depicted.

 
We next analyzed the relative representation of Gr-1+ CD11b+ myeloid cells recently described to be involved in tumor-related CD8+ T cell suppression (44). When compared with the spleen of tumor-free mice, that of tumor-bearing mice contained a higher frequency of Gr-1+ CD11b+ myeloid cells (Fig. 9D), able to prevent LACK-specific responses in vitro (data not shown). Therefore, Gr-1+ CD11b+ myeloid cells rather than CD25+ FoxP3+ cells parallel TS/A-LACK-mediated suppression.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we investigated whether DC loaded with a class II-restricted tumor-derived peptide have the capacity to prime tumor-specific CD4+ T cells and sustain the development of protective immunity. Our results indicate that this strategy confers protection against tumor challenge, but fails to cure established tumors. The differences in outcome correlate with the ability of the DC-based vaccine to elicit tumor-specific memory CD4+ T cells in the absence or in the presence of a solid tumor. Indeed, whereas peptide (LACK)-loaded DC favor the establishment of LACK-specific memory T cells in tumor-free mice, both DC-induced T cell priming and persistence are impaired in TS/A-LACK tumor-bearing mice.

In the absence of tumors, a significant fraction of I-Ad/LACK+ T cells acquired a memory phenotype in response to DC-LACK by day 6, as shown by the appearance of CD44high cells and of LACK-specific IL-2- and/or IFN-{gamma}-secreting cells. Among CD44high memory cells, a fraction preserved the surface expression of CD62L, which is typical of central memory T cells (CD44highCD62Lhigh), whereas others down-regulated this LN-homing molecule, which is typical effector T cells (CD44highCD62Llow) (36, 37, 38). Two (Figs. 3 and 4) and 3 (data not shown) wk after vaccination, LACK-specific CD44high memory cells expressing high levels of CD62L and mainly capable of IL-2 secretion (central memory-like) were present in draining and nondraining LN. In contrast, vaccination in the presence of tumors induced only suboptimal numbers of LACK-specific CD44high memory cells and cytokine-secreting cells by day 6, and memory cells were no longer found 15 days after vaccination. Thus, CD4+-directed DC vaccination primes effector-like T cells (CD44highCD62Llow, IFN-{gamma} producing), possibly able to relocate to nonlymphoid tissues, and central memory-like T cells (CD44highCD62Lhigh, IL-2 producing) able to redistribute and persist within the peripheral LN mostly in preventing settings. In the presence of tumors, priming is instead reduced, and the few primed cells disappear later on. We favor the idea that the growing tumor inhibits optimal T cell priming rather than sequestering primed T cells to the tumor-reactive LN and the tumor itself. Indeed, the frequency and total number of LACK-specific T cells in tumor-draining LN were comparable in control and vaccinated tumor-bearing mice. Furthermore, priming of LACK-specific T cells by DC-LACK vaccination (defined by CD69 up-regulation) in TS/A-LACK tumor-bearing mice was inhibited as early as 24 h after vaccine injection (data not shown). Thus, an active mechanism of suppression appears to inhibit T cell priming. We speculate that at later times, Ag re-encounter in tumor-distal LN might favor terminal differentiation of the cells, increasing their susceptibility to death-promoting signals (45, 46), relocation to nonlymphoid tissue, and exhaustion (a schematic representation of the proposed scenario is provided in Fig. 10). Because depletion of CD4+ T cells abolished DC-LACK-mediated protection, whereas the adoptive transfer of CD4+ central memory-like T cells from DC-vaccinated mice to naive animals was sufficient to delay tumor growth (our unpublished data), we speculate that the ability of tumors to impede the development of CD4+ T cell memory strongly correlates with the failure of DC-based vaccination to cause tumor rejection.


Figure 10
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  		FIGURE 10. Schematic representation of the immunogenic competence of CD4-directed DC vaccination in the absence and in the presence of tumor bulk. A, LACK-specific CD4 T cells redistribute within all lymphoid organs in the absence of tumor disease (tumor free). B, Prophylactic vaccination with peptide-loaded DC primes effector-like CD4+ T cells capable of nonlymphoid tissue homing, and central memory-like T lymphocytes with lymphoid tissue tropism. C, In the presence of TS/A-LACK tumors, effector-like CD4+ T cells are mostly primed, and cells relocate to the spleen (not depicted) and the tumor, but fail to recirculate within the lymphoid compartment. D, In the presence of tumors, peptide-loaded DC vaccination elicits lower numbers of effector-like CD4+ T cells, and primed T cells are induced to further differentiate upon Ag encounter in tumor-draining LN, possibly accounting for exhaustion of both natural and vaccine-induced antitumor CD4+ T cell responses.

 
How does the tumor limit the development of vaccine-induced T cell memory? Results reported in this study suggest that the tumor might operate at different stages, both at the time of T cell priming and during the establishment of central T cell memory (Fig. 10). In our experiments, the presence of TS/A-LACK tumors reduced the ability of DC-LACK to expand LACK-specific T cells in the LN distal to the site of tumor growth. This occurred in the absence of measurable metastatic disease (data not shown), suggesting that the suppressive activity relies on a soluble factor/s or a regulatory population able to move within the lymphoid compartment. Suppression was specific for the tumor-associated LACK Ag and required the presence of LACK-expressing tumors. Indeed, adoptively transferred OVA-specific DO11.10 TCR Tg T cells responded to comparable extents to DC-OVA (Fig. 8) or OVA/CFA vaccination (our unpublished results) in naive and TS/A-LACK tumor-bearing mice, and DC-LACK vaccination failed to prime LACK-specific memory cells in TS/A-LACK, but not in TS/A tumor-bearing mice. To better understand the origin of suppression, we have analyzed the relative representation and function of distinct population of cells able to inhibit Ag-specific and polyclonal T cell responses (40, 41, 44, 47). Flow cytometry analysis revealed comparable frequencies of CD4+CD25+ FoxP3+ (Fig. 9) and Glucocorticoid-induced TNFR-Related (GITR) protein (data not shown) T cells in the LN and spleen of control, TS/A, and TS/A-LACK tumor-bearing mice, suggesting that these cells are not enriched for in the presence of TS/A-LACK tumors. Furthermore, Ab-mediated depletion of CD25+ cells did not ameliorate vaccine (DC-LACK)-induced T cell responses in mice bearing TS/A-LACK tumors, at difference with results obtained in other models (42, 43, 48, 49, 50, 51). Thus, CD4+CD25+ FoxP3+ cells do not appear to mediate suppression in this model. Nevertheless, it is still possible that LACK-specific T cells endowed with suppressive activity are represented at frequencies below the limit of detection of the assays used in this study. In contrast to CD4+CD25+ FoxP3+ T cells, Gr1+/CD11b+ myeloid cells were increased in frequency and total number in the spleen of TS/A-LACK mice when compared with tumor-free mice, possibly in response to IFN-{gamma} secreted by tumor-primed LACK-specific CD4+ T cells. Gr1+/CD11b+ were also present in tumor-draining and nondraining LN, but at extremely low frequency, which limited their quantification (data not shown). Although Gr1+/CD11b+ myeloid cells have been described to suppress mainly CD8+ T cells (52), their ability to inhibit CD4+ T cells has also been reported (53). In preliminary experiments, we have seen that CD11b+ cells purified from tumor-bearing mice better suppress DC-LACK-mediated T cell responses in vitro when compared with CD11b+ cells purified from tumor-free control mice (our unpublished data). However, administration of sildenafil, a phosphodiesterase-5 inhibitor able to down-regulate arginase 1 and NO synthase-2 expression, thereby reducing the suppressive machinery of CD11b+/Gr-1+ myeloid-derived suppressor cells in other mouse models (54), failed to restore DC-LACK-mediated CD4+ T cell responses (our unpublished data). Thus, although systemic distribution, suppressive abilities, and relative representation of these cells render them suitable candidates for the observed immunosuppression, their role in the observed Ag-restricted inhibition of DC-mediated CD4+ T cell priming remains to be directly demonstrated.

In addition to CD4+CD25+ FoxP3+ T cells and Gr1+/CD11b+ myeloid cells, several mechanisms of Ag-directed and specific immune suppression were described in a variety of clinical settings (55). These include CD4 and CD8 double-negative regulatory T cells (56, 57), CD4+ CTLs (58), and both conventional and Qa-1-restricted CD8+-suppressive T cells able to target either CD4+ T cells (59, 60) or Ag-bearing DC (61, 62). We are performing in vivo depletions and/or passive transfers of individual subsets to determine whether any of these candidate populations play a role in hampering CD4+ T cell-directed DC vaccination. Furthermore, because IFN-{gamma} can inhibit T cell trafficking and priming (63, 64), we are investigating the possibility that the small number of LACK-specific IFN-{gamma}-secreting CD4+ T cells found in tumor-distal LN at the time of vaccination inhibits the priming of resident naive LACK-specific T cells.

Although active mechanisms of suppression are likely to be responsible for reduced priming in the context of tumor disease, Ag persistence (the growing tumor) might hamper systemic recirculation/persistence of memory cells. This has also been described in the case of chronic viral infection (65, 66). We speculate that central memory cells possibly escaping inhibitory mechanisms and primed in tumor-bearing mice are further induced to differentiate into effector/effector memory T cells upon Ag re-encounter in the tumor-draining LN, and programmed to leave the lymphoid compartment. As a consequence, tumor-specific systemic T cell memory fails to develop, thus limiting protective responses (Fig. 10).

In conclusion, the findings reported in this study, together with others (24, 67) and our previous observation (25), support the notion that tumors limit the efficacy of vaccination, and the development of both vaccine- and naturally occurring tumor-specific CD4+ T cell responses. Because proper CD4+ T cell help is needed for the development and persistence of protective responses (16, 17, 68, 69, 70), its impairment might account at least in part for the limited efficacy of DC vaccination in cancer patients (71). Further studies will be needed to design strategies able to bypass tumor-specific inhibitory mechanisms. Nevertheless, in light of these findings, and of the possibility that similar circumstances are found in cancer patients, it is reasonable to propose that vaccination should be provided at the time of diagnosis, to increase the number of effector T cells, and after tumor resection, in the absence of active disease, to favor the development of systemic, central memory-like T cell responses, which might be critical for protection against recurrent metastatic disease.


   Acknowledgments
 
We thank all of the members of the Cancer Immunotherapy and Gene Therapy Program of DIBIT/San Raffaele Scientific Institute for discussion.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 grants from the Associazione Italiana Ricerca sul Cancro, Compagnia San Paolo Istituto Mobiliare Italiano, Italian Ministry of Education (Ministero dell’Istruzione, dell’Università e della Ricerca (FIRB-RBNE017) and the European Community (contract LSHC-CT-2005-018914 "ATTACK"). Back

2 Current address: Immunomodulation and Immunotherapy, Institut de Génétique Moléculaire de Montpellier, 1919, Route de Mende, 34293 Montpellier CEDEX 5. Back

3 Current address: Institute for Research in Biomedicine, Via Vincenzo Vela 6, Bellinzona CH-6500 Switzerland. Back

4 Address correspondence and reprint requests to Dr. Anna Mondino, Cancer Immunotherapy and Gene Therapy Program, San Raffaele Scientific Institute, Via Olgettina, 58, 20132 Milan, Italy. E-mail address: anna.mondino{at}hsr.it Back

5 Abbreviations used in this paper: LACK, Leishmania receptor for activated C kinase; DC, dendritic cell; LN, lymph node; Tg, transgenic. Back

Received for publication February 7, 2007. Accepted for publication June 21, 2007.


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