Primary CD8⁺ T-Cell Response to Soluble Ovalbumin Is Improved by Chloroquine Treatment In Vivo^▿

Mice.

Female C57BL/6J mice (H-2^b) were obtained from Charles River, Calco, Italy, and maintained at the Istituto Superiore di Sanità according to the institutional guidelines. The T-cell-receptor-transgenic mouse line OT-I, expressing a T-cell receptor recognizing an H-2^b-restricted OVA_257-264 epitope, SIINFEKL, was kindly supplied by M. Bellone (San Raffaele Scientific Institute, Milan, Italy). For all experiments, mice between the ages of 6 and 12 weeks were used.

DCs.

DCs from spleens of naïve mice were purified as described previously (51). Briefly, spleen fragments were digested for 25 min at room temperature with collagenase and DNase (Sigma). EDTA (5 mM, pH 7.2; Sigma) was added for an additional 5 min to allow disruption of DC-T-cell complexes. After 2 h of incubation at 37°C in tissue culture-treated dishes, nonadherent cells were removed by gentle pipetting and the adherent cells were cultured overnight in DC medium (RPMI 1640 containing 10% fetal bovine serum, 4 mM l-glutamine, 50 μM 2-mercaptoethanol, and 10 ng/ml of granulocyte-macrophage colony-stimulating factor) (Peprotech, United Kingdom). DCs were purified by immunomagnetic bead cell sorting using anti-CD11c-conjugated magnetic beads (N418 clone; Miltenyi Biotec) according to the manufacturer's instructions. The purity of the enriched CD11c⁺ cell preparations was usually in the range between 85 and 90%.

Mouse immunization. (i) With free soluble protein.

Mice were injected intravenously (i.v.) with different concentrations of soluble OVA and treated subcutaneously (s.c.) with 800 μg of chloroquine 2 h before and 6 h after priming.

(ii) With alum-OVA.

Mice were immunized once by intraperitoneal (i.p.) injection of 200 μg of OVA protein adsorbed onto aluminum hydroxide adjuvant (Alum; Sigma). These mice were treated s.c. with 800 μg of chloroquine 2 h before and 6 h after priming.

(iii) With OVA-loaded DCs (OVA-DCs).

Splenic DCs (5 × 10⁶/ml) were pretreated with 20 μM of chloroquine (Sigma Chemical Co.) for 30 min or medium alone, followed by the addition of soluble OVA (3 to 5 mg/ml; grade V; Sigma) or 1 μg/ml of SIINFEKL peptide. Alternatively, DCs were pretreated with chloroquine, with 30 ng/ml of phorbol myristate acetate (PMA), or with both chloroquine and PMA, followed by the addition of soluble OVA. After 1 h of incubation at 37°C, extensive washes, and a 2-h chase, in the continuous presence or absence of chloroquine, 5 × 10⁵ cells were used to immunize mice i.v. Mice receiving chloroquine-treated OVA-DCs were treated s.c. with 800 μg of chloroquine 2 h before and 6 h after priming.

Cross-presentation assays in vitro.

OT-I cells were isolated from the spleen and lymph nodes of OT-I mice and further enriched for CD8⁺ T cells by treatment with monoclonal antibodies (MAbs) to CD4 (GK1.5) and major histocompatibility complex (MHC) class II (TIB120), followed by sheep anti-mouse and sheep anti-rat Dynabeads (Dynal A.S., Oslo, Norway). The final preparations from the lymphoid organs contained 75 to 85% CD8⁺ T cells. For presentation of soluble OVA, purified DCs were suspended in serum-free RPMI at a concentration of 4 × 10⁶ cells/ml and preincubated for 30 min at 37°C with or without different concentrations of chloroquine (2 μM, 6.5 μM, and 20 μM), followed by the addition of OVA (0.5 mg/ml) or 1 μg/ml of SIINFEKL peptide. After 1 h of incubation at 37°C, the cells were washed, added to microtiter plates, and coincubated with 10⁵ OT-I cells, in the continued presence or absence of chloroquine. The cells were incubated for 72 h, and 1 μCi/well [³H]thymidine (Amersham Biosciences, United Kingdom) was added 12 to 15 h before harvesting. Data are shown as mean cpm of triplicate wells minus mean cpm of corresponding control wells in the absence of antigen.

In vivo proliferation assays.

For analysis of in vivo proliferation, the enriched OT-I cells were labeled with 5- (and 6-)carboxyfluorescein diacetate, succinimidyl ester (CFSE), as previously described (32). Briefly, semipurified OT-I cells, as described above, were resuspended in PBS containing 0.1% bovine serum albumin (Sigma) at 10⁷ cells/ml and incubated with CFSE (Molecular Probes) at 5 μM for 10 min at 37°C. Cells were subsequently washed in PBS and then transferred via tail vein injection to C57BL/6 mice (2 × 10⁶ cells/mouse) that were subsequently primed 1 day later with different concentrations of soluble OVA or OVA-DCs. Three days after priming, these mice were euthanized and cells from pooled axillary, mediastinal, and inguinal lymph nodes were isolated and stained with anti-CD8-phycoerythrin (PE) for flow cytometry analysis.

In vitro cytotoxic assays.

Spleens were removed surgically 9 days after priming and prepared as a single-cell suspension. Splenocytes were stimulated in vitro for 5 days with a 2:1 ratio of splenic APCs taken from naïve mice that were pulsed for 90 min with 0.1 mM SIINFEKL peptide and then washed and gamma irradiated. Effector cells were then assayed for cytotoxic activity on ⁵¹Cr-labeled EL4 target cells pulsed or not with SIINFEKL peptide at the indicated effector/target ratios. The amount of ⁵¹Cr released was determined by gamma counting, and the percent specific lysis was calculated from triplicate samples as follows: [(experimental cpm − spontaneous cpm)/(maximal cpm − spontaneous cpm)] × 100. Spontaneous release was determined from target cells incubated in the absence of effector cells and was <5% in all experiments.

Detection of OVA-specific antibodies.

Serum was collected from individual mice on day 14 after immunization with alum-OVA, and anti-OVA antibody titers were determined by enzyme-linked immunosorbent assay. Briefly, 96-well plates (Nunc-Immuno) were coated by overnight incubation at 4°C with 100 μl of PBS containing OVA at 40 μg/ml. Plates were blocked with 1% bovine serum albumin in PBS for 2 h, and serial twofold dilutions of serum samples in PBS were added to the wells. After a 2-h incubation, plates were then washed with PBS containing 0.05% Tween 20 and incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (IgG), IgG1, or IgG2a antibodies (Southern Biotechnology Associates). After three additional washes, the plates were then incubated with p-nitrophenyl phosphate (Sigma) for 1 h at room temperature. Absorbance was read at 405 nm with a microplate reader (Bio-Rad).

Virus challenge.

The recombinant influenza A virus WSN/OVA-I, which expresses the SIINFEKL epitope, was previously generated by reverse genetics (50) and grown on Madin-Darby canine kidney cells. Mice were anesthetized with 2,2,2-tribromomethanol (Avertin) before intranasal challenge with 8 × 10⁵ PFU of WSN/OVA-I virus and euthanized 7 days postinfection.

Intracellular IFN-γ staining.

Cells derived from spleens and pooled mediastinal lymph nodes (MLN) of infected mice were cultured for 2 h in 96-well U-bottomed plates at 37°C in the presence or absence of 10 μM OVA_257-264 peptide (20). Brefeldin A (10 μg/ml) was added to each well, and the cells were incubated for an additional 4 h. The responder cells were then washed twice in brefeldin A-containing PBS (PBS-brefeldin A), treated with MAb to the Fc-γIII/II receptor to block nonspecific antibodies, and stained with a rat anti-mouse CD8-fluorescein isothiocyanate (FITC) MAb (Pharmingen). The cells were then washed again with PBS-brefeldin A, fixed in 1% formaldehyde in PBS for 20 min., washed in PBS, placed in 0.5% saponin (Sigma) in PBS for 20 min, and incubated with a rat anti-mouse IFN-γ-PE MAb (Pharmingen) or a rat IgG1-PE control MAb. Samples were then examined by FACSCalibur flow cytometry.

Tetramer staining.

The MHC class I peptide tetramer, SIINFEKL/K^b, conjugated to PE, was synthesized by Proimmune Limited (Oxford, United Kingdom). Bulk splenocytes (∼5 × 10⁶) were stained with PE-conjugated SIINFEKL/K^b tetramer, an FITC-conjugated anti-CD62L antibody (Pharmingen), and a tricolor-labeled anti-CD8 antibody (Caltag) for 30 min on ice (20). Samples were examined by FACSCalibur flow cytometry, and in the analysis CD8⁺ T cells were selected and the data plotted as tetramer (PE) versus CD62L (FITC), as indicated.

Statistical analysis.

Comparisons between experimental groups were made by using the two-tailed Student t test. P < 0.05 was considered significant.

Chloroquine improves cross-presentation of soluble OVA to naïve OT-I cells in vitro and in vivo.

Freshly isolated spleen DCs are induced to mature through overnight culture in vitro, so that they acquire the capacity to process and present newly encountered antigens (54). Chloroquine and NH₄Cl have been shown to inhibit the intravesicular acidification and marked proteolysis that occur in partially and fully mature DCs (1, 22) and also in nonprofessional APCs (i.e., T2 cells deficient in transporter associated with antigen processing) (40). Here, we first assessed the effect of chloroquine treatment on the capacity of splenic DCs to process and present exogenous soluble OVA by using an in vitro proliferation assay of OVA-specific transgenic CD8⁺ T cells (OT-I cells). The presence of low concentrations of chloroquine, 6.5 μM and 2 μM, throughout the duration of the proliferative assay improved the cross-presentation to naïve T cells (Fig. 1A). Chloroquine treatment did not affect the peptide presentation of splenic DCs loaded with the SIINFEKL peptide (OVA_257-264) to CD8⁺ T cells at these low concentrations. By contrast, the highest dose of chloroquine (20 μM) showed an inhibitory effect on T-cell proliferation in both systems, as cell numbers similar to those of the non-chloroquine-treated cells in the absence of antigen were observed. Thus, chloroquine treatment improved the cross-presentation of antigens in maturing DCs, otherwise susceptible to rapid endosomal protease degradation, sustained by the acidic pH (1).

We then examined the efficiency of cross-presentation of soluble OVA in vivo in the presence of chloroquine. In vivo cross-presentation of a soluble antigen that is not cell associated is more strictly dose related (35). It has been reported that very small amounts of cell-associated OVA are able to stimulate OT-I cells in vivo (29). On the other hand, free soluble OVA is presented much less efficiently than cell-associated OVA to OT-I cells. In our system, CFSE-labeled OT-I cells were transferred into recipient mice treated 1 day later with a single i.v. injection of various concentrations of OVA diluted in PBS and in the presence or absence of chloroquine. The proliferative responses of these cells derived from the lymph nodes were analyzed 3 days after adoptive transfer by flow cytometry (Fig. 1B). An evident in vivo OT-I proliferative response was seen in the lymphoid organs of mice given 200 μg of OVA, whereas low levels of proliferation were seen in mice given the lowest concentration (2 μg of OVA), independently from the chloroquine treatment. In mice given an intermediate dose (20 μg OVA), those receiving chloroquine during priming showed a higher proliferation of CFSE-labeled OT-I cells than did the untreated mice (P < 0.05), thus confirming the ability of chloroquine treatment to improve cross-presentation of soluble antigens to naïve CD8⁺ T cells in vivo.

Chloroquine improves induction of OVA-specific CTLs upon alum-OVA immunization in vivo.

Then we wondered whether chloroquine was capable of improving cross-priming of not only an enriched population of transferred OT-I cells but also naïve OVA-specific cytotoxic T-lymphocyte (CTL) precursors in vivo. It has been previously shown that adjuvants are required for priming CTLs with native OVA and some particulate forms of OVA. In particular, OVA in complete Freund's adjuvant (27), OVA incorporated into immunostimulating complex adjuvant (21) or into liposomes (10), or OVA adsorbed onto alum and mixed with a saponin surfactant (QS-21) (36) primed CTLs in vivo. There is very little evidence that aluminum adjuvants generate MHC class I cytotoxic T cells (30). Alum alone is a poor inducer of Th1 cellular immune responses and stimulates the production of IgE and IgG1 antibodies in mice, which is consistent with a dominant profile of Th2 lymphocyte immune response (19). Here, we examined if chloroquine treatment could improve the induction of CTLs specific for the immunodominant epitope SIINFEKL, by a single inoculation of mice with alum-OVA. To this end, spleen cells from mice primed with alum-OVA 9 days earlier were cultured with irradiated peptide-loaded syngeneic cells for 5 days and then assayed for cytolytic activity. As shown in Fig. 2A, only mice immunized in the presence of chloroquine generated detectable OVA-specific CTLs, whereas those immunized in the absence of the drug did not (P < 0.01 at the effector/target ratio of 100:1). Although the increase in antigen-specific CTL activity was not substantially high, it was nonetheless relevant, considering the poor induction of CTLs by alum-based adjuvants. We also tested the primed mice for the presence of OVA-specific IgG antibodies: in both treated and untreated groups, the antibody levels were comparable (P > 0.05), and IgG1 was the dominant isotype, independently of the chloroquine treatment (Fig. 2B).

Chloroquine treatment during priming with alum-OVA improves the induction of memory CD8⁺ T cells.

It was necessary to determine whether the increased numbers of primed CTLs due to the combination of a single dose of alum-OVA and chloroquine treatment have the capacity to develop effective long-term memory T cells. The magnitude of a secondary immune response correlates with the size of memory T cells induced by the cross-presented antigen during priming (28, 34). To this end, mice were vaccinated with alum-OVA in the presence or absence of chloroquine and 8 weeks later we elicited a recall response by boosting them with WSN/OVA-I virus bearing the T-cell epitope SIINFEKL in the stalk region of the viral neuraminidase (50). Seven days after the boost, we determined the proportion of OVA-specific CD8⁺ T lymphocytes in the pooled MLN and the spleens that were functional by intracellular IFN-γ staining. Approximately 7.6% and 6.6% of CD8⁺ T cells in MLN and bulk splenocytes, respectively, of chloroquine-treated mice produced IFN-γ after stimulation with OVA peptide, versus approximately 2.8% and 2.5% of CD8⁺ T cells from the untreated mice (P < 0.01) (Fig. 3A). Lower levels of antigen-specific CD8⁺ T cells were found at this time of infection in the unprimed mice. Moreover, lymphocytes that were not stimulated with peptide showed background staining similar to that of the isotype control immunoglobulin (<1%; data not shown). The higher proportion of CD8⁺ T cells in bulk splenocytes of chloroquine-treated mice that showed as MHC class I-SIINFEKL tetramer positive and CD62L^Lo (Fig. 3B) further indicates that priming of mice in the presence of this drug induces larger CD8⁺ T-cell memory responses.

Overall, these results show that concurrent immunization of mice with alum-OVA and chloroquine treatment supports higher CD8⁺ T-cell expansion of primary effectors and thus a large population of memory CD8⁺ T cells that undergo a rapid expansion and recruitment following pulmonary infection with the recombinant influenza virus.

Chloroquine improves cross-priming via its effect on DCs.

To ascertain that the improvement in cross-priming through chloroquine in vivo was related to chloroquine's capacity to increase cross-presentation by DCs, as observed in our studies in vitro (Fig. 1A), CFSE-labeled transgenic OT-I cells were transferred into C57BL/6 mice primed 1 day later with OVA-DCs, which had been treated with chloroquine. In particular, splenic DCs cultured overnight were pretreated in vitro with chloroquine or medium alone for 30 min followed by the addition of 3 mg of soluble OVA or 1 μg of SIINFEKL peptide for 1 h and extensively washed. After a 2-hour chase, chloroquine-treated OVA-DCs were injected into mice, who received chloroquine s.c. (in order to guarantee a continuous presence of chloroquine during antigen processing in vivo), whereas the untreated OVA-DCs were injected into chloroquine-untreated mice. Three days after priming, recipients were euthanized and the lymphoid organs were analyzed by flow cytometry. As shown in Fig. 4, OT-I cells proliferated more in the lymphoid organs of chloroquine-treated mice than in untreated mice which had received OVA-DCs (P < 0.05), thus confirming that chloroquine treatment improves in vivo cross-priming of naïve CD8⁺ T cells by maturing DCs. It is improbable that cross-priming was due to contaminating soluble OVA, because OVA-DCs were extensively washed and chased before the immunization procedures and because cross-priming by soluble antigens (including OVA) generally requires high antigen concentrations (Fig. 1B) (29). Similar percentages of proliferating OT-I cells induced upon immunization with SIINFEKL-DCs were measured independently of the chloroquine treatment.

To further corroborate the finding that chloroquine was capable of improving the primary antigen-specific response of CTL precursors in vivo by boosting the cross-presentation capacity of DCs (Fig. 1A), we tested the additive effect of PMA and chloroquine because the former has already been demonstrated to improve cross-presentation (37, 38) and to function as a nonspecific Rho GTPase activator capable of promoting endocytosis and antigen presentation by murine DCs (47). The presence of OVA-specific CTL effector cells in the spleens was determined on day 9 by ⁵¹Cr release assay, after one round of restimulation in vitro in the presence of peptide-loaded syngeneic cells (Fig. 5). Groups of mice injected with SIINFEKL-pulsed DCs were used in these assays to make sure that the chloroquine treatment in vivo did not affect the ability of DCs to present the synthetic peptide. Mice treated with chloroquine and receiving chloroquine-treated OVA-DCs efficiently induced SIINFEKL-specific CTLs, whereas the untreated group of mice did not. Furthermore, the combined use of chloroquine and PMA in vitro and subsequent injection in chloroquine-treated mice determined an additive effect that more closely correlates the functional role of the chloroquine with the reduced antigen proteolysis and enhanced cross-presentation which occurs in maturing DCs. As expected, the injection of splenic DCs stimulated with PMA alone in the presence of OVA gave lower levels of antigen-specific CTL induction than those obtained with the combined use of both the drugs, thus being similar to levels obtained by the use of chloroquine alone (Fig. 5).