CD8⁺ T Cell-Dependent Elimination of Dendritic Cells In Vivo Limits the Induction of Antitumor Immunity

Mice

C57BL/6 mice were from breeding pairs originally obtained from The Jackson Laboratory (Bar Harbor, ME). Strain 318 mice (15), transgenic for a TCR specific for H-2 D^b plus fragment 33–41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV_33–41),³ were kindly provided by Dr. H. Pircher (Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany). The B6Aa⁰/Aa⁰ MHC class II^−/− mice (16) were supplied by Dr. H. Bluethmann (Hoffmann-LaRoche, Basel, Switzerland). All mice were maintained at the Biomedical Research Unit of the Wellington School of Medicine by brother × sister mating; in vivo experimental protocols were approved by the Wellington School of Medicine Animal Ethics Committee and were performed according to institutional guidelines.

Tumor cell line

LL-LCMV is a derivative of the Lewis lung carcinoma LLTC (C57BL/6, H-2^b) that has been modified to express a minigene encoding LCMV_33–41 under the control of a CMV promoter (17).

In vitro culture media and reagents

Unless otherwise stated, all cultures were maintained in complete medium (CM) comprising of IMDM with 2 mM glutamine, 1% penicillin-streptomycin, 5 × 10⁻⁵ M 2-ME, and 5% FBS (all from Life Technologies, Auckland, New Zealand). The synthetic peptides LCMV_33–41 (KAVYNFATM) and OVA_257–264 (SIINFEKL) were from Chiron Mimotopes (Clayton, Australia).

Culture of bone marrow-derived DC

Bone marrow cells from C57BL/6 mice or B6Aa⁰/Aa⁰ mice were cultured at 4 × 10⁵ cells/ml in CM containing 20 ng/ml IL-4 and 20 ng/ml GM-CSF as described previously (18). Cultures were provided fresh CM and cytokines every 3 days and incubated at 37°C until the time of assay (6–8 days). Cultures typically contained 70–100% DC as determined by fluorescent staining with the anti-CD11c Ab N418. DC (1 × 10⁶ cells/ml) were loaded with peptide Ag by incubation at 37°C in CM containing 10 μM synthetic peptide for 2 h and then were washed three times with IMDM to remove excess peptide.

DC migration assay

DC were labeled with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) by incubation in PBS containing 1 μM CFSE for 10 min at 37°C before one wash in five volumes of ice-cold PBS and two washes with IMDM. Unless otherwise stated, mice were injected with 1 × 10⁶ CFSE⁺ DC by s.c. injection in the distal forelimb (volar aspect). At the indicated times, both axillary and brachial lymph nodes were removed from immunized mice and incubated in 2.4 mg/ml collagenase type II (Life Technologies) and 1 mg/ml DNase I (Sigma, St. Louis, MO) for 90 min at 37°C. The tissue was then disrupted by aspiration through an 18-gauge needle. The cell suspension was sieved through gauze, washed in PBS, and resuspended for flow cytometric analysis in PBS containing 2% FBS and 0.01% sodium azide.

Lymph node suspensions were analyzed using a FACSort and CellQuest software (both from Becton Dickinson, Mountain View, CA). The region containing DC was identified on the basis of forward/side light scatter profile. No CFSE⁺ cells were found outside this region. In initial experiments, the total number of DC within the DLN was calculated as follows: % CFSE⁺ cells in the DC region × % of DLN cells in the DC region × number cells in DLN; data are expressed as average number of DC within DLN for each experimental group. Because it was observed over repeated experiments that the percentage of CFSE⁺ cells in the DC region was always proportional to the absolute number of CFSE⁺ cells across the experimental groups, data were expressed as average percentage of CFSE⁺ cells thereafter. Only events falling within the gated DC region were collected and stored (>250,000 events for each DLN suspension).

Frozen sections (8 μm) were prepared from the axillary lymph nodes of animals injected with CFSE⁺ DC 20 h earlier. The sections were stained with hematoxylin and eosin and analyzed by standard and fluorescence microscopy. CD8⁺ T cells are depleted by one i.v. injection of 500 μg purified 2.43 mAb 24 h before DC immunization.

Ab staining

Ab staining was in PBS containing 2% FBS and 0.01% sodium azide. The anti-FcγRII mAb 2.4G2 was used at 10 μg/ml to inhibit nonspecific staining. Anti-MHC class II (3JP) and anti-CD11c (N418) were affinity purified from culture supernatants using protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden), conjugated to biotin as described (19), and revealed using streptavidin-PE (PharMingen, San Diego, CA). Instrument compensation was set in each experiment using single color-stained samples.

Adoptive transfer and DC immunization

Pooled lymph node cell suspensions were prepared from strain 318 mice, and the percentage of T cells expressing the transgenic TCR was determined on one sample of cells by staining with anti-Vα2 and anti-Vβ8.1, 8.2 Abs and by FACS analysis. The cells were then labeled by incubation in 1.25 μM CFSE (Molecular Probes) in PBS for 10 min at room temperature at a cell concentration of 2 × 10⁷ cells/ml. The cells were pelleted by centrifugation in the presence of 50% FCS, washed two times in CM, and then washed again in IMDM. Suspensions containing 5 × 10⁶ Vα2⁺Vβ8⁺ cells were transferred into C57BL/6 recipients by i.v. injection into the tail vein. One day after adoptive transfer, the mice were immunized s.c. with 10⁵ DC. After 66 h, the draining axillary and brachial lymph nodes were removed, teased through gauze to prepare single-cell suspensions, and analyzed by FACS.

Tumor immunity assay

Groups of C57BL/6 mice (n = 5) were immunized by s.c. injection into the left flank with 10⁵ DC. Secondary immunizations were by s.c. injection into the contralateral flank 7 days later. One week after the last immunization, all animals were challenged with 10⁶ LL-LCMV tumor cells injected s.c. into the left flank. Mice were monitored for tumor growth every 3–4 days, and tumor size for each group was calculated as the mean of the products of bisecting diameters (±SE). Measurements were terminated for each group when the first animal developed a tumor in excess of 200 mm².

CFSE⁺ DC injected s.c. are detected in the DLN

We examined the migration of injected DC to the secondary lymphoid organs of mice. DC were cultured from bone marrow precursors in the presence of GM-CSF and IL-4 for 7 days and labeled with CFSE, an amine-reactive dye that can be retained in live cells for many generations. DC were administered s.c. into the distal forelimb of mice without any deliberate preincubation with Ag. As can be seen in Fig. 1⇓, 20 h after injection CFSE⁺ cells could be detected in the DLN by fluorescence microscopy on frozen tissue sections and were located in the paracortical region of the lymph node. Analysis of DLN cell preparations by flow cytometry also revealed the presence of CFSE⁺ cells in the DLN (Fig. 2⇓). No CFSE⁺ cells could be detected in the non-DLN. In addition, CFSE⁺ cells could not be detected in the DLN if the DC were heat killed before injection. These results imply that the CFSE⁺ cells had reached the DLN via an active migratory process.

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FIGURE 1.

DC injected s.c. migrate to the paracortex of the DLN. Mice were injected s.c. with 5 × 10⁵ CFSE⁺ DC in the distal forelimb, and axillary and brachial lymph nodes were removed for histological analysis 20 h after injection. Frozen tissue sections of the DLN were prepared and stained with hematoxylin and eosin. CFSE⁺ cells were detected by fluorescence microscopy. A, Low-power magnification with a representative CFSE⁺ cell indicated (arrowhead). B, High-power magnification showing three CFSE⁺ cells.

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FIGURE 2.

DC that have migrated to the DLN can be detected by FACS analysis. Mice were injected s.c. with 5 × 10⁵ CFSE⁺ DC in the distal forelimb, and axillary and brachial lymph nodes were removed for FACS analysis 20 h after injection. Data were collected in a gate encompassing only large cells as based on the known size of cultured DC. No CFSE⁺ cells were found outside this gate. A, Results are shown for a noninjected animal, an animal injected with CFSE⁺ DC (DLN and contralateral nodes), and an animal that was injected with CFSE⁺ DC that had previously been heated to 80°C for 10 min. B, DLN from C57BL/6 mice injected with either C57BL/6 DC (MHC class II^+/+ DC) or B6Aa⁰/Aa⁰ DC (MHC class II^−/− DC). DLN cell suspensions were stained with anti-MHC class II to determine MHC class II expression on CFSE⁺ cells.

The possibility that the CFSE⁺ cells found in the DLN were in fact endogenous cells that had acquired CFSE from the injected cells was examined by injecting MHC class II^−/− DC and then using MHC class II expression to distinguish the endogenous from the injected DC. C57BL/6 mice were injected with CFSE⁺ DC cultured from either C57BL/6 mice or MHC class II^−/− B6Aa⁰/Aa⁰ mice. MHC class II expression was then examined on CFSE⁺ cells in the DLN. As shown in Fig. 2⇑B, MHC class II expression could be demonstrated on CFSE⁺ cells obtained from mice that had received C57BL/6 DC. In contrast, no expression of MHC class II was found on CFSE⁺ cells from mice that received MHC class II^−/− DC. We conclude that the CFSE⁺ cells detected in the DLN were the same DC that had been injected peripherally and that these cells had actively migrated from the site of injection to the DLN.

The proportion of CFSE⁺ DC that reached the DLN and their kinetics of appearance were also examined. As shown in Fig. 3⇓A, the absolute number of CFSE⁺ DC found within the DLN represented only a small proportion (∼0.1%) of the original number of DC injected. The injected DC appeared in the DLN within 12 h of injection, reaching maximum numbers before 48 h. Considerable numbers of CFSE⁺ DC could still be demonstrated in the DLN at 172 h postinjection (Fig. 3⇓B), the latest time point examined.

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FIGURE 3.

Quantitation and kinetics of DC migration to the DLN. A, Groups of mice were given s.c. injections in both distal forelimbs with the indicated numbers of CFSE⁺ DC; DLN were removed for FACS analysis 20 h later. Each group contained three animals with each injected forelimb treated as an independent event. B, Mice were injected with 10⁶ CFSE⁺ DC, and the DLN were removed for FACS analysis at the indicated time points. Three animals were analyzed at each time point with each injected forelimb treated independently (n = 6). Data are presented as the mean percentage of DC collected in a gate encompassing only large cells (±SE), as outlined in Materials and Methods.

In summary, CFSE⁺ DC can be demonstrated to actively migrate from the site of s.c. injection to the DLN and to persist in vivo in excess of 7 days from the time of injection.

DC loaded with MHC class I-binding peptide Ag are cleared from the DLN

Next, we sought to determine the fate of Ag-loaded DC after s.c. injection into mice. CFSE⁺ DC were loaded with LCMV_33–41, an MHC class I-binding peptide from the LCMV glycoprotein, and injected into the forelimbs of C57BL/6 recipients. The numbers of Ag-loaded DC in the DLN were compared with the numbers in the DLN of mice that received DC not loaded with Ag. As can be seen in Fig. 4⇓A, loading with Ag had no effect on the number of CFSE⁺ DC recovered from the DLN at 16 h after injection. By 72 h, a small reduction in the number of DC in the DLN was observed in the experiment shown, but this reduction was not always observed in repeated experiments. In contrast, when the number of CD8⁺ T cells specific for the Ag presented on DC was increased as in TCR transgenic recipients, a significant and highly reproducible reduction in the number of Ag-loaded DC in the DLN was observed 72 h after injection. No reduction was apparent at 16 h. Depletion of CD8⁺ T cells from TCR transgenic recipients before DC injection prevented the reduction in number of Ag-loaded DC in the DLN (Fig. 4⇓B). These results suggest that activation of TCR transgenic CD8⁺ T cells to cytotoxic effector cells is associated with the disappearance of DC expressing specific Ag from the DLN. DC clearance occurs between 16 and 72 h after injection, a period consistent with the acquisition of effector function by CD8⁺ T cells as reported by other investigators (20, 21). The minimal clearance of Ag-loaded DC demonstrated in the DLN of naive, non-TCR transgenic mice probably reflects the smaller number of Ag-specific T cells that become activated in these mice and hence the lower number of interactions that result in DC clearance.

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FIGURE 4.

DC loaded with MHC class I-binding peptide Ag are cleared from the DLN. Groups of C57BL/6 or TCR transgenic mice were injected s.c. in the forelimb with either 10⁶ CFSE⁺ DC or 10⁶ CFSE⁺ DC that had been loaded with the LCMV_33–41 peptide recognized by the transgenic TCR. A, DLN were removed for FACS analysis 16 or 72 h after CFSE⁺ DC injection. B, TCR transgenic recipients were depleted of CD8⁺ T cells or were left untreated before injection with CFSE⁺ DC. DLN were removed for FACS analysis 72 h later. All data are presented as in Fig. 3⇑B.

Clearance of Ag-loaded DC is accelerated in the presence of an Ag-specific immune response

Next, we next investigated the fate of Ag-loaded DC in the context of an ongoing immune response. To this end, CFSE⁺ DC loaded with the MHC class I-binding peptide LCMV_33–41 were administered to mice that had been injected s.c. in the flank 7 days before with 10⁵ DC alone or with 10⁵ DC loaded with the same LCMV_33–41 peptide. The 7-day interval was chosen because our previous studies have shown that Ag-specific T cell expansion is maximal at this time (22); also, resistance to challenge with LCMV_33–41-expressing tumors can be demonstrated (17). In addition, “memory” recipients were also used. These animals had received LCMV_33–41-loaded DC 6 mo before and had subsequently rejected a challenge with tumor cells expressing the LCMV_33–41 epitope. All mice received the LCMV_33–41-loaded, CFSE⁺ DC by s.c. injection into the anterior forelimb. As can be seen in Fig. 5⇓, similar numbers of CFSE⁺ DC were observed in the DLN of mice that were either previously immunized with DC without Ag or in those of mice that had received no previous treatment. In contrast, a large reduction in the number of CFSE⁺ DC was observed in the DLN of animals immunized with LCMV_33–41 peptide-loaded DC 7 days earlier. The reduction was clearly detectable as early as 20 h after injection, suggesting that it was related to the presence of peptide-specific effector cells in the recipient mice. Similar results were also obtained with MHC class II^−/− DC (data not shown), indicating that CD4⁺ T cells were not involved in DC elimination. A decrease in the number of CFSE⁺ DC was also observed in the DLN of mice from the “memory” group. However, in this latter case the decrease in CFSE⁺ DC was observed only at 72 h and not at 20 h after in vivo injection. This result suggests that the reduction in the number of CFSE⁺ DC was related to the acquisition of effector activity by pre-existing memory CD8⁺ T cells in the recipient mice.

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FIGURE 5.

Clearance of Ag-loaded DC is accelerated in the presence of an Ag-specific immune response. The presence of CFSE⁺, LCMV_33–41 peptide-loaded DC was monitored in the DLN of mice that had been immunized 1 wk before with 10⁵ LCMV_33–41-loaded DC injected s.c. in the flank or with 10⁵ DC not loaded with Ag or in mice that had received no previous treatment. A fourth group, termed the “memory” group, was also assessed. This group had been immunized with 10⁵ LCMV_33–41 peptide-loaded DC 6 mo before and had also successfully rejected a challenge with 10⁶ LL-LCMV tumor cells 7 days after immunization. All groups were injected s.c. with 10⁶ CFSE⁺, LCMV_33–41 peptide-loaded DC in each forelimb. Three animals from each group were analyzed at the indicated time points after CFSE⁺ DC administration. Data are presented as in Fig. 3⇑B.

Taken together, the data presented above indicate that the disappearance of CFSE⁺ DC from the DLN of recipient mice is associated with the presence or the development of effector CTL. We suggest that in the course of an immune response, Ag-loaded DC become targets for CTL activity and are eliminated from the lymph node.

CTL-mediated clearance of DC from the lymph node impairs induction of further immune responses

We wished to establish whether elimination of Ag-loaded DC during an immune response impacts upon T cell activation initiated by the same DC. As an in vivo readout of T cell activation, we examined the proliferation of CFSE-labeled, LCMV_33–41-specific CD8⁺ T cells adoptively transferred into syngeneic recipients. Proliferation of CFSE⁺ T cells can be monitored in the DLN of immunized mice by flow cytometry and is detected as progressive halving of cellular fluorescence with every cell division completed (23). Thus, we examined the specific proliferation induced by DC that were or were not the targets of a pre-existing immune response. Mice were preimmunized s.c. in the flank with DC + OVA_257–264 peptide to elicit an OVA-specific CTL immune response or with DC only as a control. One week later, both groups of mice received an adoptive transfer of CFSE⁺ LCMV_33–41-specific T cells and were immunized s.c. in the forelimb with DC loaded with both the OVA_257–264 and LCMV_33–41 peptides. These mice were used to examine the proliferation of LCMV_33–41-specific T cells. In addition, separate mice that had also been immunized in the flank with DC + OVA_257–264 or with DC only received CFSE⁺ DC that had been loaded with both the OVA_257–264 and LCMV_33–41 peptides by s.c. injection in the forelimb. These latter mice were used to evaluate the numbers of CFSE⁺ DC in the DLN. As shown in Fig. 6⇓, OVA_257–264 + LCMV_33–41-loaded DC were rapidly eliminated in mice that had previously been immunized with DC + OVA_257–264, most likely due to the induced OVA-specific CTL response. In contrast, a considerable proportion of OVA_257–264 + LCMV_33–41-loaded DC could be demonstrated in the DLN of mice previously immunized with DC only. Fig. 6⇓ also shows the proliferative response of LCMV_33–41-specific T cells in mice that had received the same DC immunizations. In mice in which DC could be demonstrated in the DLN, LCMV_33–41-specific T cells underwent several cycles of cell division, indicating the onset of an active immune response. In contrast, in mice in which DC had disappeared from the DLN, only limited proliferation of LCMV_33–41-specific T cells could be demonstrated. We conclude from these results that CTL-mediated elimination of DC may have a profound impact on T cell immune responses and may prevent the initiation of immune responses to other Ags that are simultaneously presented on the same DC.

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FIGURE 6.

CTL-mediated clearance of Ag-loaded DC from the DLN limits the induction of further immune responses. Groups of mice were immunized s.c. in the flank with either 10⁵ OVA_257–264-loaded DC or with 10⁵ DC alone. All mice received a second injection of DC loaded with OVA_257–264 and LCMV_33–41 s.c. in the forelimb. Survival of the DC administered with the latter injection and their induction of a T cell response were examined. Left panel, CFSE⁺ DC simultaneously loaded with OVA_257–264 and LCMV_33–41 are eliminated in OVA-immunized mice. Data were collected 66 h after injection of CFSE⁺ DC and are presented as in Fig. 3⇑B. Right panel, Adoptively transferred CFSE⁺ TCR transgenic cells specific for LCMV_33–41 fail to divide in response to specific peptide presented on DC if the DC are eliminated by an existing CTL response. Data were collected 66 h after DC immunization; only CFSE⁺, Vα2⁺ Vβ8.1,8.2⁺ events are shown. FACS profiles shown are of one representative mouse of the six in each group.

CTL-mediated clearance of tumor Ag-loaded DC from the lymph node impairs antitumor immune responses

We wished to extend our findings by examining whether elimination of Ag-loaded DC during the induction of an antitumor immune response has a significant negative impact on antitumor immunity. For these studies we used the tumor cell line LL-LCMV, a Lewis lung carcinoma expressing the LCMV_33–41 epitope (17). All mice were preimmunized s.c. in the flank with DC loaded with OVA_257–264 to induce OVA_257–264-specific CTL responses. One week later, mice received a second s.c. immunization in the opposite flank with MHC class II^−/− DC that were loaded with both OVA_257–264 and the “tumor Ag” LCMV_33–41 or with MHC class II^−/− DC loaded with the “tumor Ag” LCMV_33–41 only. MHC class II^−/− DC were used in this experiment to rule out any effects mediated by CD4⁺ T cells. Similar results were obtained when MHC class II^+/+ DC were used (data not shown), because DC are eliminated by pre-existing CTL responses regardless of MHC class II expression. DC presenting OVA_257–264 and tumor Ag became targets of the anti-OVA_257–264 CTL response and were eliminated (data not shown). In contrast, DC presenting tumor Ag alone remained unaffected. One week after the second DC immunization, mice were challenged with LL-LCMV, and tumor size was measured. In Fig. 7⇓, we show that DC presenting both OVA_257–264 and LCMV_33–41 were less effective at inducing antitumor immunity than DC presenting the LCMV_33–41 Ag alone were. This reduction in efficacy was most likely due to the rapid clearance of OVA_257–264-loaded DC by OVA_257–264-specific CTL, which prevented the productive initiation of an LCMV_33–41-specific immune response. However, a limited degree of antitumor immunity was reproducibly induced by OVA_257–264-loaded DC in these experiments, suggesting that the DC were able to induce some degree of T cell activation before being eliminated. Alternatively, DC-associated Ag had been released and taken up by other APC, allowing a specific immune response to be initiated.

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FIGURE 7.

CTL-mediated clearance of tumor Ag-loaded DC from the DLN impairs the generation of antitumor immune responses. Two groups of mice (n = 5) were immunized with 10⁵ OVA_257–264 peptide-loaded DC by s.c. injection in the flank, and then 1 wk later, one group was immunized with LCMV_33–41 peptide-loaded MHC class II^−/− DC in the contralateral flank, and the second group was immunized with MHC class II^−/− DC loaded with both LCMV_33–41 and OVA_257–264 peptides. After 7 days, both of these groups and an additional control group were challenged with 10⁶ LL-LCMV tumor cells presenting the LCMV_33–41 epitope. Mice were monitored for tumor growth every 3–4 days, and tumor size for each group was calculated as the mean of the products of bisecting diameters (±SE). Measurements were terminated for each group when the first animal developed a tumor in excess of 200 mm².