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Listeriolysin O Expressed in a Bacterial Vaccine Suppresses CD4⁺CD25^high Regulatory T Cell Function In Vivo¹

Josianne Nitcheu-Tefit^*, Ming-Shen Dai^*,, Rebecca J. Critchley-Thorne^2,*, Francisco Ramirez-Jimenez, Man Xu^*,, Sophie Conchon^¶,||, Nicolas Ferry^¶,||, Hans J. Stauss and Georges Vassaux^3,*,¶,||

^* Centre for Molecular Oncology, Institute of Cancer, Queen Mary’s School of Medicine and Dentistry, London, United Kingdom; Department of Immunology and Molecular Pathology, Royal Free Hospital, London, United Kingdom; Department of Pathology, Chongqing University of Medical Sciences, Chongqing, China; Division of Haematology/Oncology, Tri-Service General Hospital, National Defense Medical Centre, Taipei, Taiwan; ^¶ Institut National de la Santé et de la Recherche Médicale Centre d’Investigation Clinique-04, Universite de Nantes, Nantes Atlantique Universites, Centre Hospitalier de l’Université (CHU) Hotel Dieu, Nantes, France; and ^|| Institut des Maladies de l’Appareil Digestif, CHU Hotel Dieu, Nantes, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺CD25^high regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8⁺ depletion—but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44^highCD62L^low CD8⁺ effector memory T cells over CD44^highCD62L^high central memory T cells. Unexpectedly, CD4⁺ depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4⁺ cells suppressed the CD8⁺ T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4⁺CD25^high regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4⁺CD25^high expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Bacteria encoding tumor Ags, used as gene or protein delivery systems, have been extensively described in the literature to produce significant and sometimes spectacular antitumor effects in preclinical models (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The general concept is that the bacteria’s pathogen-associated molecular patterns act as adjuvants to mount an effective immune response against bacteria-encoded tumor Ags. These pathogen-associated molecular patterns may include among other bacterial components LPS involving TLR4 (14, 15), CpG motifs of bacterial DNA recognized by TLR9 (16), and flagellin, sensed by TLR5 (17).

However, the application of this technology to humans is limited by safety concerns (the bacteria are generally associated with pathologies) and pathogenic genes or proteins present in the bacteria may not be required for the therapeutic effect. To address these concerns, our approach has involved the use of a nonpathogenic strain of Escherichia coli that has been engineered to express a minimal number of relevant genes "borrowed" from pathogenic bacteria, necessary and sufficient to confer antitumor activity. So far, the most efficient strain tested is a recombinant E. coli that coexpresses the Ag of interest and listeriolysin O (LLO)⁴ from Listeria (E. coli LLO), a member of the pore-forming cytolysins capable of binding and perforating phagosomal membranes at low pH (18, 19). Upon s.c. injection, it is understood that this bacteria is internalized by APCs, taken into the phagosome/lysosome where lysis of the bacterium occurs. Through the pore-forming action of LLO, the cytoplasmic contents of the bacteria can then escape into the cytosol and thereby be processed by the proteasome. In vitro, this LLO-mediated process has been shown to improve MHC class I presentation of the OVA H2-K^b-restricted epitope SIINFEKL by mouse macrophages (20), mouse bone-marrow-derived dendritic cells (BMDCs) (6), the HLA-A2-restricted MART1_27–35 epitope (21), and the immunodominant epitope of the influenza matrix protein (22) by human monocytes-derived DCs. In vivo, s.c. injection of E. coli LLO expressing the model chicken OVA Ag (E. coli LLO/OVA) has been shown to trigger a very strong antitumor response against the highly aggressive B16/OVA melanoma cell line (6) (B16-OVA). The antitumor effect of E. coli LLO/OVA was Ag specific and far superior to that of E. coli expressing OVA only (E. coli OVA). Interestingly, the therapeutic effect does not require live bacteria as it is also observed when the bacteria are fixed with paraformaldehyde before injection (6, 13).

Considering that improved MHC class I presentation of antigenic peptides is unlikely to be the sole mechanism responsible for the striking difference in efficacy between E. coli OVA or E. coli LLO/OVA vaccines, the aim of the present study was to determine how the expression of LLO in the bacteria can turn a marginally active bacterial vaccine (E. coli OVA) into a potent antitumor vaccine (E. coli LLO/OVA).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

Six- to 8-wk-old female C57BL/6J mice (20–25 g) were obtained from Harlan Breeders and kept in a germfree environment with irradiated food and acidified water ad libitum. Experiments were conducted after appropriate ethical approval and licensing was obtained in accordance with the U.K. "Guidance on the Operation of Animals (Scientific Procedure) Act 1986" (HMSO, London, U.K.).

Peptides

OVA_257–264 SIINFEKL peptide was synthesized at >95% purity. The peptide 130 (NAPYLPSCL) is derived from WT1 protein and was used as an irrelevant peptide. Peptides were synthesized by the Peptide Synthesis Laboratories (Cancer Research U.K.).

Cell lines

RMA-S cells, derived from RMA (K^bD^b) (23), were cultured in complete RPMI 1640 medium containing 10% FCS. B16 and B16/OVA (the B16 melanoma cell line transfected with the OVA gene; CRUK, London, U.K.), were maintained in DMEM containing 10% FCS supplemented with 400 mg/ml G418.

E. coli immunization/treatment protocols and tumor challenge

C57BL/6 mice were vaccinated s.c. with several injections doses of 10⁸ of either E. coli expressing OVA (E. coli OVA) or E. coli coexpressing LLO and OVA (E. coli/LLO/OVA) at a 1-wk interval. Control groups were immunized with E. coli, E. coli expressing LLO (E. coli/LLO), or PBS (see Refs. 6 and 20 for details of the bacteria). Mice were challenged 1 wk after the last vaccination by injection of 5 x 10⁵ B16-OVA cells in the tail vain. Following injection, B16/OVA cells home to the lungs where the cells form multiple nodules. Experiments were terminated and the whole cohort was culled when the first animal became visibly unwell (24–28 days following tumor challenge), then the tumor growth was analyzed by counting nodules in the lungs. For survival experiments, challenged mice in a group were individually monitored until signs of sickness were observed. In treatment experiments, mice were first injected i.v. with B16/OVA tumor cells and treated with s.c. injections of the various bacteria 8 and 15 days later.

In vitro CTL stimulation and CTL assay

Stimulating cells were prepared by activating syngeneic spleen cells at 10⁶ cells/ml with 25 mg/ml LPS and (Sigma-Aldrich) and 7 mg/ml dextran sulfate (Sigma-Aldrich). The cells were collected 3 days after activation, irradiated (3000 rad), and loaded with the peptides.

RMA-S cells were temperature induced for MHC class I expression at 26°C overnight, and binding of the peptides to H-2K^b and H-2D^b was allowed to proceed for 2 h at 37°C with the optimal concentration of the peptide determined in pilot experiments. Effectors cells from vaccinated mice were then mixed with stimulators and cultured for 5–6 days before analyzed in standard ⁵¹Cr-release assays (23). Specific killing was calculated as ((experimental release – spontaneous release)/(maximum release – spontaneous release)) x 100.

Isolation of BMDCs

BMDCs were prepared as previously described (6). For loading, 1x10⁸ of either bacteria was added to 1x10⁶ BMDCs in a volume of 1 ml in polypropylene tubes in RPMI 1640 medium supplemented with 10% FCS. After 1 h of incubation, Ag-pulsing medium was decanted by several washes before the BMDCs were used for assay.

Abs and in vivo depletion of T cell subsets

The anti-CD4 (GK1.5), anti-CD8 (YTS 169.4), and PLTY-1 (isotype control) mAbs were purified from relevant hybridomas (Cancer Research U.K.). Anti-CD25 mAb (clone PC61) and its isotype control (rat IgG1) were purchased from BioExpress. On days 0 and 7, C57BL/6 mice were vaccinated s.c. with 10⁸ of either bacterium. Depletion of CD4⁺ T cells was achieved at the time of T cell priming by i.p. administration of 300 µg of GK1.5 on days –5 and 10. CD8⁺ T cells were depleted by in vivo administration of 400 µg of YTS 169.4-depleting mAbs on days 10 and 17. Control mice were treated with same doses of relevant mAb isotype controls or PBS. Preparatory experiments revealed that YTS 169.4 or GK1.5 mAbs totally depleted CD8⁺ or CD4⁺ population, respectively, in the spleen, lymph nodes (LN), and peripheral blood 4 days following injection, as measured by flow cytometric analysis, while PLTY-1 mAb control did not deplete either CD4⁺ or CD8⁺ T cells. To deplete CD25⁺ cells, a total of 400 µg of PC61 Ab was injected i.p. on day –1 before vaccinations. Optimal conditions of depletion of these T cell subsets were determined in a preparatory experiment and were shown to totally delete CD25⁺ cells in peripheral blood and significantly diminish the percentage of CD25⁺ in the spleen and LN for at least 10 days following the injection.

ELISPOT assay for IFN- production

Ninety-six-well ELISPOT plates (Millipore) were coated with 100 µl/well of 15 µg/ml purified anti-mouse IFN- mAb (BD Biosciences) overnight at 4°C. Plates were washed five times with PBS before addition of 8x10⁵ splenocytes in triplicate wells and 10 µM peptide. Con A was used as a positive control. After 20 h of incubation at 37°C in 5% CO₂, plates were developed by incubating with 50 µl/well of biotinylated anti-IFN- (BD Biosciences) at 1 µg/ml in PBS for 2 h at 37°C. Streptavidin alkaline phosphatase (100 µl; Caltag Laboratories) was added to each well after five washes and incubated for 1 h at room temperature (RT). The plate was developed using 100 µl of alkaline phosphatase-conjugate substrate (BioRad). Spots were counted by an automated ELISPOT reader and a response was considered positive when spot numbers in triplicate assays in the presence of the specific peptide significantly exceeded the cutoff value, corresponding to the number of nonspecific spots in the presence of irrelevant peptide.

Abs and pentamers and flow cytometric analysis

Anti-CD3 FITC, Anti-CD4 PerCP, anti-CD8 PerCP, anti-CD62L allophycocyanin, and anti-CD44 PE were purchased from BD Biosciences. PE-conjugated H-2K^b/SIINFEKL pentamers were purchased from Proimmune. The Mouse Regulatory T Cell Staining kit was purchased from eBioscience. Single-cell suspension obtained from spleen and LNs were treated in ammonium chloride potassium (ACK) buffer to lyse erythrocytes, washed three times, and resuspended in PBS containing 3% FCS (FACS buffer). The cells were blocked for nonspecific binding with anti-FcRII before being incubated with optimal concentration of appropriate mAbs for 30 min on ice, then washed and resuspended in FACS buffer.

For pentamer staining, the cells were first labeled with the pentamer for 10 min at RT, washed once, then incubated with optimal concentration of anti-CD8. Pentamer staining was analyzed by gating on CD8⁺ cells. For regulatory T cell staining with the staining set (eBioscience), cells were treated according to the manufacturer’s instructions. Data were collected using a FACSCalibur cytometer (BD Biosciences) and analyzed using CellQuestPro software (BD Biosciences).

CD4⁺CD25^– and CD4⁺CD25⁺ T cells selection, proliferation, and regulatory cell culture assays

Single-cell suspensions were obtained from the spleens of vaccinated mice. CD4⁺ T cells were negatively selected and fractionated into CD4⁺CD25^– and CD4⁺CD25⁺ subsets by magnetic Ab cell sorting (MACS; Miltenyi Biotec), using PE-labeled anti-CD25 mAb followed by anti-PE microbeads, according to the manufacturer’s instructions. The purity of cells was checked by FACS analysis and >90% of CD4⁺ cells were shown to be either CD25^– or CD25⁺. For proliferation/regulatory assays, 100 x 10³ responder cells (CD4⁺CD25^– or CD8⁺ T cells) or CD4⁺CD25⁺ regulatory T cells were cultured in RPMI 1640 medium containing 10% FCS, 50 µM 2-ME with 0.5 µg/ml anti-CD3 mAb (purified anti-CD3; BD Pharmingen) in the presence of 200 10³ naive irradiated splenocytes. In the MLR, various amounts of regulatory T (Treg) cells were added to 100 x 10³ responder cells. The cells were cultured for 4 days and proliferation was measured by adding 1 µCi of [³H]thymidine (Amersham) to each well for the last 18 h of culture period. The cells were harvested and the thymidine incorporated was determined.

Cytokine assays

IFN-, IL-2, IL-4, and IL-10 were quantitated in the sera, in culture supernatants of splenocytes, and in E. coli-activated BMDCs cultures. Mice received two s.c. injections of the various bacteria, then sera and splenocytes were collected 1 wk after the boost injection. Splenocytes were left unstimulated or restimulated with 10 mM SIINFEKL for 3 days. BMDCs were infected or not with the various E. coli and cultured for 48 h. The DuoSet ELISA system (R&D Systems) was used to measure cytokine production according to the manufacturer’s protocol.

Statistics

Statistical analysis was performed using Prism (GraphPad Software). Dual comparisons were made using the unpaired Student t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Coexpression of LLO in the E. coli vaccines allows the expansion of OVA-specific CD8⁺ T cells

Animals were given several s.c. injections of 10⁸ formaldehyde- fixed E. coli, E. coli LLO, E. coli OVA, or E. coli LLO/OVA or PBS, and enumeration of OVA-specific CD8⁺ T lymphocytes was performed on splenocytes collected 1 wk after each last injection. Plots depicting percentages of CD8⁺, pentamer-positive cells gated on live lymphocytes are shown in Fig. 1a. Mice receiving E. coli OVA vaccines showed little but not significant increase of SIINFEKL/H2-K^b specific CD8⁺ T lymphocytes above the background levels observed in E. coli, E. coli LLO, or PBS-vaccinated animals (1 ± 0.2 vs 0.5 ± 0.2% of total splenocytes, respectively). By contrast, E. coli LLO/OVA-vaccinated mice induced significant levels of SIINFEKL/H2-K^b-specific CD8⁺ T cells and the highest percentage and absolute numbers (4% of total splenocytes, 3.8 ± 1.2 x 10⁶ cells) was achieved following one boost injection (Fig. 1b). As it has been shown that LLO induces apoptosis of infected cells and activated lymphocytes (24, 25), it is likely that multiple boosts may lead to the death of these cells, altering the response. "Prime-boost" vaccines strategies combining naked DNA and E. coli may help to amplify Ag-specific immune responses. Thus, vaccines combining LLO and OVA allow the activation of OVA-specific CD8⁺ T cells, confirming the previously reported (6) importance of the expression of LLO in the bacterial vaccine.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. E. coli LLO/OVA vaccination allows the activation of OVA-specific CD8+T cells as measured by multimeric MHC-peptide analyses. Mice received several injections of bacteria at a 1-wk interval. Splenocytes were then collected 7 days after each last injection and stained with the SIINFEKL/H-2Kb pentamers. a, Representative experiment depicting percentages of CD8+, pentamer-positive cells gated on live lymphocytes are shown. b, The percent and absolute numbers of SIINFEKL/H-2Kb-specific T cells in the spleen (mean ± SD) after a single vaccination, one or two boost injections are shown. ***, p < 0.001.

To examine the ability of the vaccines to control tumor, animals received two injections of each of the E. coli vaccines at 1-wk interval followed by i.v. challenge of 5 x 10⁵ B16 cells expressing OVA (B16/OVA). In this model, B16/OVA cells home to the lungs where the cells form multiple tumor nodules. When the first mice began to show signs of sickness (typically 24–26 days following B16/OVA challenge), the whole cohort was culled and the tumor load was assessed by counting tumor nodules in the lungs.

Fig. 2 shows that the lungs of animals vaccinated with E. coli or E. coli LLO were heavily colonized by tumors (82 ± 11 and 86 ± 12 nodules, respectively). The burden was modestly but significantly reduced in mice vaccinated with E. coli OVA (60 ± 12 per set of lungs, p = 0.01 vs E. coli) and dramatically reduced in E. coli LLO/OVA-vaccinated animals (5 ± 2, p < 0.0001 vs E. coli LLO). The time to lethal tumor burden in E. coli OVA-vaccinated mice was extended from 39 to 48 days (compare with E. coli-immunized mice) while E. coli LLO/OVA vaccine induced complete protection and survival over 200 days in 70% of mice (data not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 2. E. coli LLO/OVA vaccine inhibits lungs metastasis induced by i.v. injection of B16 cells expressing OVA. C57BL/6 mice were given two s.c. injections of bacteria at a 1-wk interval, then were challenged a week after the boost injection by tail-vein injection of 5 x 105 B16/OVA cells. Animals were sacrificed when the first mouse showed signs of disease (typically 24–28 days following tumor challenge) and the tumor growth was assessed by counting tumor nodules in the lungs. Results are expressed as mean ± SD. The experiments presented are representative of at least three experiments involving six animals per groups. *, p = 0.01; ***, p < 0.001.

E. coli LLO/OVA vaccination induce high-avidity CTLs

Because OVA-specific CD8⁺ T cell expressing the immunodominant T cell epitope OVA_257–264 SIINFEKL are clearly critical to the antitumor effect leading to the in vivo rejection of B16/OVA tumor (6, 26), the presence of SIINFEKL-specific CTLs was tested in mice in the prophylactic vaccination setting. To characterize the avidity of the cytotoxic responses, splenocytes from vaccinated mice were restimulated in vitro with SIINFEKL-loaded LPS-stimulated spleen cells and CTL activity was measured 6 days later using SIINFEKL-loaded RMA-S and B16/OVA cells as targets. The results are summarized in Fig. 3a. Splenocytes from E. coli LLO/OVA-vaccinated animals showed a strong response against SIINFEKL-loaded RMA-S cells and B16/OVA tumor cells (>75% killing at the highest E:T ratio), suggesting that the CTLs are of high avidity. By contrast, weak CTL responses against SIINFEKL-loaded RMA-S cells and B16/OVA tumors (<25% killing at the highest E:T ratio) were detected in splenocytes cultures from E. coli OVA-vaccinated mice, while no cytotoxic activity was found in splenocyte cultures from E. coli or E. coli LLO control mice. ELISPOT assay measuring IFN- secretion by T cells confirmed the requirement of LLO to activate T cell function (Fig. 3b): only E. coli LLO/OVA SIINFEKL-stimulated splenocytes induced significant IFN- production (250 ± 84 spots/well vs 48 ± 8 or 50 ± 16 spots/well in E. coli and E. coli OVA-vaccinated animals, respectively; p = 0.004).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. E. coli LLO/OVA vaccination generates high avidity CTL responses. a, C57BL/6 mice received two s.c. injections of bacteria at a 1-wk interval. Splenocytes prepared from spleens collected 1 wk later were restimulated in vitro with LPS-stimulated spleen cells loaded with the OVA-restricted class I SIINFEKL peptide. Cytotoxicity was measured 6 days later by 51Cr-release assay against SIINFEKL or irrelevant peptide-loaded RMAS cells as well as B16/OVA and the parental B16 tumor cell lines. Specific 51Cr release was calculated as described in Materials and Methods. Results from individual mice are plotted against the E:T ratio, and are representative of at least two experiments. b, Mice received two s.c. injections of bacteria at a 1-week interval. Splenocytes were collected after the boost injection and incubated overnight with the SIINFEKL peptide, and ELISPOT assay was used to measure IFN- secretion. Results are expressed as mean ± SD. Splenocytes from mice that received vaccines coexpressing LLO and OVA induced a significant strong response (**, p = 0.0043). The experiments presented are representative of two separate experiments involving three animals per group.

In this study, we investigate the ability of the E. coli vaccines to program memory responses. Central memory T cells (T_CM) and T_EM can be differentiated by the relative expression of CD62L on the CD44^high population (27, 28). We used these markers to analyze the accumulation of CD4 and CD8 T_CM and T_EM in the spleen and inguinal lymph nodes of vaccinated animals. Results showed that the proportions of total memory phenotype were similar between E. coli OVA and E. coli LLO/OVA. Both vaccines generated a pool of memory T cells biased toward effector responses (T_EM) compared with PBS-injected mice (see Table I).

To assess the relative contribution of T cell subsets in tumor protection, CD8⁺ as well as CD4⁺ T cells were depleted in vivo in the vaccination model. CD4⁺ T cell depletion was conducted at the stage of T cell priming (on days 5 and 10 after the first vaccination). CD8⁺ T cells were depleted on days 10 and 17 after bacterial injections. CD8⁺ T cells depletion reversed the protection induced by E. coli LLO/OVA vaccine as well as the modest but significant protection observed upon E. coli OVA vaccination, demonstrating the essential role of CD8⁺ T cells in tumor protection (Fig. 4a). CD4⁺ T cells depletion at the stage of T cell priming had no impact on E. coli LLO/OVA vaccination, suggesting that CD8⁺ T cell priming in vivo can occur in the absence of CD4⁺ T cell help. Unexpectedly, CD4⁺ T depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4⁺ T cells inhibited the cells mediating the antitumor response. Moreover, mice receiving combined treatments with depleting Abs against CD4 and CD8 T cells showed progressive tumor growth, confirming that the antitumor activity unmasked by CD4⁺ T cell depletion is dependent on the presence of CD8⁺ T cells (Fig. 4a).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. CD8+ T cells are the key mediators for tumor protection and Treg cells prevent their activation in mice receiving the E. coli OVA vaccines. a, Mice received two s.c. injections of bacteria on days 0 and 7. Depletions were conducted by i.p. injection of GK1.5-depleting mAb (anti CD4) at the stage of T cell priming (on days –5 and 10) in combination or not with i.p. injections of YTS169.4 (anti-CD8) on days 10 and 17. As a control, an irrelevant mAb control (PYLT-1) was used. Mice were challenged on day 14 by tail-vein injection of 5 x 105 B16/OVA cells and sacrificed when the first mouse showed signs of disease (typically 24–28 days following tumor challenge), then the tumor load was assessed in the lungs. Results are expressed as mean ± SD. The experiments presented are representative of two experiments involving six animals per groups; ***, p < 0.0001. b, Depletion of CD25+ cells was conducted by i.p. injection of the PC61 mAb on day –1 before s.c. bacterial vaccinations on days 0 and 7. As a control, an irrelevant mAb (RatIGg1) was used. Mice were challenged on day 14 by tail-vein injection of 5 x 105 B16/OVA cells and sacrificed when the first mouse showed signs of disease (typically 24–28 days following tumor challenge), then the tumor load was assessed in the lungs. Results are expressed as mean ± SD. The experiments presented are representative of two experiments involving six animals per groups. ***, p < 0.0001. c, Depletion of CD4+ or CD25+ cells was conducted as described above, then splenocytes prepared from spleens harvested 1 wk after the boost injection was incubated overnight with the SIINFEKL peptide. ELISPOT assay was used to measure INF- secretion. The experiments presented are representative of three experiments involving three animals per groups. Results are expressed as mean ± SD. **, p = 0.0016 (GK1.5 vs mAb control treatment); **, p = 0.0023 (PC61 vs mAb control treatment).

E. coli OVA and E. coli LLO/OVA vaccines induce similar frequencies of CD4⁺CD25^high Treg cells

To investigate whether LLO expression affects Treg cell expansion, we compared the prevalence of these cells in the spleen and LNs close to the site of inoculation in vaccinated animals. No significant differences in CD4⁺CD25^high T cell frequencies were found in the spleens and inguinal LNs in all groups of mice, as assessed by FACS analysis, and Foxp3⁺ expression within this population was similar (80–100%) (Table II). (For an example of CD4⁺CD25^high T cells, see Fig. 5a).

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Flow cytometric analyses of CD4+CD25high, CD4+CD25–, and CD4+CD25+ T cells. a, Representative experiment depicting CD4+CD25high gated on live lymphocytes is shown. b, Single-cell suspensions were obtained from the spleens of vaccinated mice. CD4+ T cells were negatively selected and fractionated into CD4+CD25– and CD4+CD25+ subsets by MACS (Miltenyi Biotec), using PE-labeled anti-CD25 mAb followed by anti-PE microbeads, according to the manufacturer’s instructions. The purity of the cells was analyzed by staining with anti-CD4 and anti-CD25 Abs.

To test their functionality, Treg were purified after the vaccination regimen and yielded a CD4⁺CD25⁺ population that was >90% pure (Fig. 5b). We performed titration studies with different amounts of Treg mixed with their corresponding CD4⁺CD25^– or CD8⁺ responders (conventional T cells (T_CONV)) (10⁵/assay). Cultures were stimulated with an anti-CD3 mAb. Treg cells from each of the vaccine systems did not proliferate upon TCR stimulation (data not shown). In the MLR, T_CONV proliferation from E. coli or E. coli OVA-vaccinated animals was gradually reduced according to Treg dosing, and >50% inhibition was observed at a Treg/T_CONV ratio of 1:1 (Fig. 6a). By contrast, inhibition of proliferation of responders was dramatically reduced with Treg cells from E. coli LLO or E. coli LLO/OVA-vaccinated mice (<20% at the highest ratio). Importantly, Treg cells from the E. coli OVA vaccine system inhibited the proliferation of CD4⁺CD25^– or CD8⁺ responders from the E. coli LLO/OVA vaccine system as effectively as they inhibited their corresponding responders (Fig. 6b). Thus, animals receiving E. coli LLO or E. coli LLO/OVA vaccines had overall reduced regulatory functions compared with mice receiving E. coli or E. coli OVA vaccines, suggesting that LLO expression in the E. coli vaccines reduces/suppresses Treg cell function.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. CD4+CD25+ T cells isolated from E. coli OVA or E. coli LLO/OVA-vaccinated animals induce different levels of suppression. a, Mice received two s.c. injections of E. coli, E. coli LLO, E. coli OVA, or E. coli LLO/OVA and splenocytes prepared from spleens harvested 7 days after the boost injection were separated into CD4+CD25+ and CD4+CD25– cells. In a MLR, CD4+CD25high (variable number) were cultured with CD4+CD25– (105) T cells for 4 days in the presence of 0.5 µg/ml anti-CD3 and 2 x 105 naive irradiated splenocytes. Proliferation of responders was measured by adding [3H]thymidine for the last 18 h of culture period and inhibition of proliferation was determined. The experiments presented are representative of three independent experiments. ***, p < 0.001 E. coli vs E. coli LLO and E. coli OVA or E. coli LLO/OVA. b, Mice received two s.c. injections of E. coli OVA or E. coli LLO/OVA and splenocytes prepared from spleens harvested 7 days after the boost injection were separated into CD4+CD25+ and CD4+CD25– cells. CD8+ T cells were also purified from E. coli LLO/OVA-vaccinated animals. In a MLR, CD4+CD25+ (variable numbers) from E. coli OVA were cultured with 105 of their corresponding responders (CD4+CD25– T cells) or responders from E. coli LLO/OVA-vaccinated animals (CD4+CD25– or CD8+ T cells) for 4 days in the presence of 0.5 µg/ml anti-CD3 and 2 x 105 naive irradiated splenocytes. Proliferation of responders was measured by adding [3H]thymidine for the last 18 h of culture period and inhibition of proliferation was determined. The experiments presented are representative of two independent experiments.

Finally, we analyzed the cytokine response induced by the different vaccines in the sera, in SIINFEKL-stimulated splenocytes cultures, and in the various E. coli activated-BMDCs cultures. Results are summarized in Table IV. IL-2, IL-4, IL-10, and IFN- serum levels were just above the threshold of detection and not significantly different between any of the animals given the E. coli vaccines. IFN- levels were significantly lower in splenocyte cultures from animals that received the E. coli OVA vaccines as compare with animals that received E. coli LLO/OVA vaccines (450 ± 14 vs 841 ± 102 pg/ml). The same trend was found in BMDC cultures that were activated by E. coli and E. coli OVA as compare with BMDCs cultures that were activated by E. coli LLO and E. coli LLO/OVA, while the inverse correlation was observed for IL-10 production. Altogether, these data support the conclusion that the response to E. coli LLO/OVA vaccines is CD8 mediated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In a previous report (5), a recombinant Listeria monocytogenes strain that expresses and secretes the human papilloma virus E7 protein fused to a nonhemolytic form of LLO (Lm-LLO-E7) was shown to be effective against established E7-expressing tumors. Interestingly, no protection was observed with a strain that expresses and secretes E7 alone, not fused to LLO, (Lm-E7) and depletion of CD4⁺ or CD25⁺ cells turned Lm-E7 into an effective treatment (5). The authors found increased numbers of CD4⁺CD25^high T cells in Lm-E7-vaccinated mice compared with Lm-LLO-E7-immunized animals and no difference in Treg cell function (30). The design and modes of action of these bacterial vaccines may explain these differences. In our study, we used an E. coli strain of bacteria, a Gram-negative, while Listeria, a Gram-positive bacterium was used for the other study. In addition, recombinant Listeria are likely to reach the cytosol intact and will secrete either E7 and LLO-E7 proteins, while E. coli LLO/OVA LLO perforates the lysosomal membrane and allows the release of the bacterial contents in the cytosol (LLO is not fused to the Ag and lacks its secretion signal sequence).

In mice, the elimination of CD4⁺ suppressor T cells, using various strategies has been largely reported to enhance antitumor immunity (31, 32, 33, 34, 35, 36). Many studies have reported elevated levels of CD4⁺CD25⁺ T cells in patients with different types of cancers (37, 38, 39, 40, 41). Similarly, greater disease burden and poorer overall survival are correlated to increased Treg cells (1, 39, 42, 43). These observations have led to the development of new therapeutic strategies aiming at the elimination of Treg cells in cancer patients and, so far, a single clinical trial has been reported, involving an IL-2/diphtheria toxin conjugate to target CD25 at the surface of Tregs (44). In this context, the recombinant E. coli LLO-expressing tumor Ags provides a unique system by inducing specific cytotoxic responses and selectively inhibiting regulatory T cell function. However, it is still unexplained how LLO inhibits Treg cell function and studies are currently underway to determine the exact mechanism of this inhibition.

The E. coli vaccination system offers a number of advantages. First, the bacteria are nonpathogenic and the delivery of Ag relies on the lysis of the bacteria by APCs. The paraformaldehyde fixation kills the bacteria while retaining the antitumor activity, providing an additional safety feature. Furthermore, as the full-length cDNA of the Ag is expressed in the bacteria, the vaccine is unlikely to be restricted to a specific HLA haplotype in humans. Current work in our laboratory in the context of tumor Ags that are self Ags has demonstrated that vaccination with recombinant E. coli LLO expressing the Wilm’s tumor 1 Ag (WT-1) (45, 46) led to a significant control of WT-1-expressing tumors in C57BL/6J mice, correlated to specific CTL responses and Treg functional defect (J. Nitcheu-Tefit, M. S. Dai, and G. Vassaux, unpublished observations). Based on these results, it is possible to envisage the use of this system in humans.

	Acknowledgments

 
We thank Dr. K. Radford for helpful discussion, Prof. D. Mazier for practical help, and Sandra Peak, Sarah Scott, and Maurel Tefit for technical support.

	Disclosures

Top 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 Cancer Research U.K., the Medical Research Council, Institut National de la Santé et de la Recherche Médicale (INSERM), and by Grant 0607-3D1615-66/AO INSERM from the French National Cancer Institute (INCa).

² Current address: Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305.

³ Address correspondence and reprint requests to Dr. Georges Vassaux, Institut National de la Santé et de la Recherche Médicale CIC-04, EE 0502, 3ème étage HNB nord, Centre Hospitalier de l’Université Hotel Dieu, 1 place Alexis Ricordeau, 44035 Nantes Cedex 1, France. E-mail address: georges.vassaux{at}nantes.inserm.fr

⁴ Abbreviations used in this paper: LLO, listeriolysin O; DC, dendritic cell; BMDC, bone marrow-derived DC; LN, lymph node; Treg, regulatory T; T_EM, effector memory T; T_CM, central memory T; T_CONV, conventional T.

Received for publication January 11, 2007. Accepted for publication May 20, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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