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Impaired Ability of MHC Class II^-/- Dendritic Cells to Provide Tumor Protection is Rescued by CD40 Ligation¹

Ian F. Hermans^2,*, David S. Ritchie^2,*, Angela Daish^*, Jianping Yang^*, Marilyn R. Kehry and Franca Ronchese^3,*

^* Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington, New Zealand; and Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877

	Abstract

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The contribution of CD4⁺ T cells to dendritic cell (DC) activation and to the induction of CD8⁺ T cell responses in vivo was investigated using a model of antitumor immune responses. Immunization with peptide-loaded MHC class II-deficient (MHC class II^-/-) DC induced the activation of Ag-specific CD8⁺ T cells and their accumulation in the lymph nodes and spleens of immunized mice. The accumulation induced by MHC class II^-/- DC immunization was lower than the accumulation observed after immunization with MHC class II^+/+ DC. Similarly, immunization with peptide-loaded, MHC class II^-/- DC induced some degree of protection against tumor challenge, but this protection was lower than the protection achieved after immunization with MHC class II^+/+ DC. Incubation with a membrane-associated form of CD40 ligand resulted in the up-regulation of costimulatory molecules on MHC class II^-/- DC and fully rescued their ability to induce antitumor immunity. We conclude that CD4⁺ T cells play a critical role in the generation of antitumor immune responses through their capacity to induce the activation of DC via CD40/CD40 ligand interaction, and thus maximize CD8⁺ T cell responses.

	Introduction

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Provision of "help" by activated CD4⁺ T cells is a crucial step in the generation of CTL responses. Several studies have shown that not only is effective CTL immunity totally dependent upon help from CD4⁺ T cells (1, 2), but that help must be provided in a cognate manner, such that both the Th cells and the CD8⁺ T cells recognize Ag on the same APC (3). However, other studies, particularly those considering antiviral CTL immunity, have shown that cytotoxic responses may be elicited in the absence of CD4⁺ T cell help (4). These apparently contradictory data were reconciled by recent studies suggesting that CD4⁺ Th cell function is mediated through activation of APCs, specifically dendritic cells (DC).⁴ Activated DC up-regulate costimulatory and adhesion molecules (5, 6) and thus become able to stimulate CD8⁺ T cells to acquire cytotoxic function (7, 8, 9). The key interaction involved in APC activation is mediated by CD40 ligand (CD40L) on Th cells and CD40 on DC (5, 6, 7, 8, 9, 10). Furthermore, DC may be activated by other stimuli, such as viral infection (7, 11) or exposure to inflammatory signals including endotoxin and TNF- (5, 6). The availability of these other signals may therefore explain the variable dependence upon CD4⁺ T cell help in CTL-mediated immunity.

In the studies referenced above, the requirement for CD40 ligation and DC activation in CD8⁺ T cell responses was demonstrated using in vitro readouts of CTL-mediated killing (7, 8, 9). We wished to extend those findings to an in vivo readout of CTL activity; for this purpose, we chose to use a model of T cell-mediated tumor immunity in which CD8⁺ T cells activated through immunization with Ag-loaded DC can mediate protection to a subsequent tumor challenge. In this report we show that, in the absence of CD4⁺ T cell help, DC immunization provides incomplete protection to tumor challenge. However, optimal protection can be restored by direct stimulation of DC with CD40L before immunization.

	Materials and Methods

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Mice

C57BL/6 mice were from breeding pairs originally obtained from The Jackson Laboratory (Bar Harbor, ME). The "318" mice (12), which were transgenic for a TCR specific for H-2 D^b plus fragment 33–41 of the lymphocytic choriomeningitis virus (LCMV) glycoprotein (LCMV_33–41) were kindly provided by Dr. H. Pircher (Institute of Medical Microbiology, University of Freiburg, Freiburg, Germany). The B6Aa⁰/Aa⁰ MHC class II-deficient (MHC class II^-/-) mice (13) were kindly provided by Dr. H. Bluethmann (Hoffmann-La Roche, Basel, Switzerland). All mice were maintained at the Animal Facility of the Wellington School of Medicine by brother x sister mating. All in vivo experiments were approved by the Wellington School of Medicine Animal Ethics Committee.

Tumor cell line, in vitro culture media, and reagents

The tumor cell line LL-LCMV is a derivative of the Lewis lung carcinoma LLTC (C57BL/6, H-2^b), which has been modified to express a minigene encoding LCMV_33–41 under the control of a CMV promoter (14). Unless otherwise stated, all cultures were in IMDM (Life Technologies, Auckland, New Zealand) containing 2 mM glutamine, 1% penicillin-streptomycin, 5 x 10^-5 M 2-ME (all from Sigma, St. Louis, MO), and 5% FCS (Life Technologies). LL-LCMV was maintained in culture medium containing 0.5 mg ml^-1 G418 (Life Technologies). The synthetic peptide LCMV_33–41 (KAVYNFATM) was obtained from Chiron Mimotopes (Clayton, Australia). Cell membranes containing mouse CD40L (membrane-bound CD40L (mbCD40L)) were prepared from the Sf9 insect cell line infected by a baculovirus vector containing a CD40L gene construct as described previously (15).

FACS staining and reagents

Anti-CD11c (N418), anti-FcRII (2.4G2), anti-Vß8.1, 8.2 (KJ16.133.18), anti-I-A^b (3JP), and anti-K^b (28.13.3s), and anti-CD44 (I42/5) were affinity-purified from tissue culture supernatants and conjugated to FITC or biotin as described previously (16). Anti-V2-PE, anti-CD80-PE, and anti-CD86-FITC were obtained from PharMingen (San Diego, CA). FACS staining was performed as described previously (17).

Preparation of bone marrow-derived DC

Bone marrow cells from C57BL/6 (MHC class II^+/+) or MHC class II^-/- mice were cultured in medium containing 20 ng/ml IL-4 and 20 ng/ml GM-CSF as described previously (18). Cultures typically contained 90–100% N418⁺ cells as determined by FACS staining. In some experiments, DC were stimulated with a 1/1000 dilution of a CD40L-expressing Sf9 cell membrane preparation over the last 48 h of culture. DC were loaded with peptide by incubation in medium containing 10 µM of peptide for 2 h at 37°C and subsequently washed three times before injection.

Adoptive transfer of T cells and DC immunization

Pooled lymph node (LN) cell suspensions were prepared from strain 318 mice; the percentage of T cells expressing the transgenic TCR was determined by staining with anti-TCR V2 and anti-TCR Vß8.1, 8.2 mAb and by FACS analysis. Groups of C57BL/6 recipient mice were injected i.v. with 3–5 x 10⁶ V2⁺Vß8⁺ T cells. After 1 day, recipients were immunized by a s.c. injection in the flank with 10⁵ peptide-loaded or untreated DC in IMDM. For each experiment, a group of adoptive transfer recipients was left unmanipulated to serve as a control. Spleens and inguinal draining LNs were harvested at different times postimmunization for analysis of cellular composition by FACS.

Tumor protection assay

Groups of three to six mice were immunized by s.c. injection in the right flank with 10⁵ DC that had been loaded with LCMV_33–41 peptide or left untreated; the mice were challenged 7 days later with 1 x 10⁶ LL-LCMV tumor cells injected s.c. into the left flank as described previously (14). For some experiments, 5 x 10⁶ V2⁺Vß8⁺ T cells were adoptively transferred into each animal 1 day before DC immunization. Mice were monitored every 3–4 days, and the mean tumor size for each group was calculated as the mean product of bisecting tumor diameters.

	Results

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Ag-loaded DC induce activation and accumulation of adoptively transferred TCR transgenic T cells

The activation and accumulation of specific CD8⁺ T cells after DC immunization was examined by following the response of a population of TCR transgenic CD8⁺ T cells adoptively transferred into syngeneic recipients. Approximately 50% of CD8⁺ T cells from the 318 transgenic strain carry a transgenic V2⁺Vß8.1⁺ TCR specific for LCMV_33–41 in association with H-2D^b (12). The equivalent of 3 x 10⁶ V2⁺Vß8⁺ transgenic T cells were injected i.v. into C57BL/6 hosts; after 1 one day, the recipients were immunized with autologous DC loaded with synthetic LCMV_33–41 peptide Ag. The percentages of V2⁺Vß8⁺ cells in the LNs and spleens of immunized mice were assessed by FACS staining at different times postimmunization. The proportion of V2⁺Vß8⁺ T cells was increased 4-fold in mice immunized with Ag-loaded DC, peaking at day 5 postimmunization in the LNs and at day 7 postimmunization in the spleen (Fig. 1). Control animals that had been immunized with DC not loaded with Ag showed no alteration in percentages of V2⁺Vß8⁺ T cells. In addition, expression of the CD44 activation marker was specifically enhanced on V2⁺Vß8⁺ T cells in response to immunization with Ag-loaded DC, with elevated levels still detectable in the LN and spleen at 14 days postimmunization (Fig. 1). In contrast, no increased expression of CD44 was detected on V2⁺Vß8⁺ T cells in the LNs or spleens of mice injected with DC only. Therefore, immunization with DC loaded with MHC class I-binding peptide Ag results in the transient accumulation of activated, Ag-specific CD8⁺ T cells in the draining LNs and, later, in the spleens of immunized mice.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Accumulation and activation of V2+Vß8+ T cells after immunization with Ag-loaded DC. At 24 h after the adoptive transfer of TCR transgenic V2+Vß8+ cells, C57BL/6 mice were immunized with DC that had been loaded with Ag (•) or with DC that were left untreated (). Percentages of V2+Vß8+ cells were determined in the draining LNs (left panels) or spleens (right panels) at various times postimmunization. CD44 expression on gated V2+Vß8+ cells is displayed in the lower panels as mean fluorescence intensity (determined by three-color FACS analysis). For each timepoint, the average ± SE of two to three animals is given.

Ag-loaded MHC class II^-/- DC induce reduced accumulation of V2⁺Vß8⁺ cells as compared with MHC class II^+/+ DC

To establish whether CD8⁺ T cell activation was compromised in the absence of CD4⁺ T cell help, we examined the activation and accumulation of adoptively transferred V2⁺Vß8⁺ T cells after immunization with MHC class II^-/- DC. FACS analysis of MHC class II^+/+ and MHC class II^-/- DC before in vivo injection revealed a similar expression of costimulatory molecules (data not shown). The DC-induced accumulation of V2⁺Vß8⁺ T cells in the spleens of recipient mice was examined on day 7 postimmunization, when the response at this site is maximal. Immunization with Ag-loaded MHC class II^+/+ DC resulted in an average 2.7-fold increase in the percentage of V2⁺Vß8⁺ T cells over controls (Fig. 2). These V2⁺Vß8⁺ T cells expressed increased levels of the activation marker CD44 (data not shown). Interestingly, immunization with Ag-loaded MHC class II^-/- DC also resulted in a specific increase in the percentage of the V2⁺Vß8⁺ cells in the spleen. However, this increase was lower than the increase observed after immunization with MHC class II^+/+ DC (Fig. 2). Immunization with MHC class II^-/- DC in hosts that were also MHC class II^-/- again induced specific CD8⁺ T cell activation and accumulation (data not shown), indicating that the observed CD8⁺ T cell response was truly independent of CD4⁺ T cell help. Therefore, we conclude that optimal CD8⁺ T cell activation requires CD4⁺ T cell help, although some activation can occur in its absence.

View larger version (46K): [in this window] [in a new window]   FIGURE 2. DC-induced accumulation of V2+Vß8+ T cells is impaired in the absence of CD4+ T cell help. Percentages of V2+Vß8+ cells were determined in the spleens of C57BL/6 mice after adoptive transfer of transgenic V2+Vß8+ cells and immunization with MHC class II+/+ or MHC class II-/- DC that had been loaded with Ag (hatched bars) or left untreated (open bars). The average fold increase (± SE) in the percentage of V2+Vß8+ T cells over nonimmunized adoptive transfer controls on day 7 postimmunization is shown. Combined data from three separate experiments, each using three to five mice per group, are presented.

We have shown previously that immunization with LCMV_33–41 Ag-loaded DC induces protective immunity against challenge with the LL-LCMV tumor, which expresses the LCMV_33–41 epitope (14). Therefore, we compared the growth of LL-LCMV tumors in mice that had been immunized s.c. with Ag-loaded MHC class II^-/- DC or MHC class II^+/+ DC 7 days before tumor challenge. A representative experiment is shown in Fig. 3A; combined data from several experiments are shown in Fig. 3B. Animals immunized with Ag-loaded MHC class II^+/+ DC showed delayed tumor growth when compared with unimmunized mice (data not shown) or mice immunized with DC alone. At day 25 after tumor challenge, the average tumor sizes were 14 mm² in mice immunized with Ag-loaded MHC class II^+/+ DC and 85 mm² in mice immunized with DC alone. By contrast, animals immunized with Ag-loaded MHC class II^-/- DC exhibited an impaired antitumor immune response as demonstrated by the larger size of tumors at all times postimmunization (average size at day 25 = 53 mm²). These results indicate that the activation of CD4⁺ Th cells and hence the provision of help are required not only for the in vivo activation of CD8⁺ T cells but also for optimal antitumor activity.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. MHC class II-/- DC induce antitumor immunity less effectively than MHC class II+/+ DC. A, C57BL/6 mice were immunized with MHC class II+/+ DC that had been loaded with LCMV33–41 peptide or left untreated or immunized with LCMV33–41 peptide-loaded MHC class II-/- DC. Tumor challenge occurred 7 days after DC immunization. Mean tumor size ± SE and the numbers of animals per group that developed tumors are shown. B, Mice were immunized and challenged as described in A. Data from three separate experiments are combined; the mean tumor size ± SE on day 25 after tumor challenge is shown.

To test the hypothesis that the reduced antitumor immunity induced by immunization with MHC class II^-/- DC was due to the lack of CD4⁺ T cell help via CD40/CD40L, MHC class II^-/- DC were incubated in vitro with CD40L before in vivo injection. mbCD40L, which is derived from a CD40L-expressing insect cell line (15), was used to stimulate MHC class II^-/- DC over the last 48 h of culture. DC stimulated with mbCD40L showed increased expression of CD80 and CD86 relative to nonstimulated cultures (Fig. 4). Expression of murine DC Ag, CD11c, and MHC class I were not altered following treatment. In control experiments, treating DC with a membrane preparation from a mock-infected insect cell line produced no alteration of DC activation markers (data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Surface marker expression on MHC class II-/- DC stimulated with CD40L. The expression of CD11c, CD80, CD86, MHC class I, and MHC class II was analyzed on unstimulated DC or DC treated with mbCD40L over the last 48 h of culture. Thick line, Ab staining as indicated; thin line, nonstained control. In the lower two panels, the control and MHC class II profiles are superimposed.

View larger version (44K): [in this window] [in a new window]   FIGURE 5. MHC class II-/- DC stimulated with CD40L in vitro are as effective as MHC class II+/+ DC at inducing antitumor immunity. A, C57BL/6 mice received an adoptive transfer of TCR transgenic T cells and were then immunized with LCMV33–41 peptide-loaded MHC class II+/+ DC or with LCMV33–41 peptide-loaded MHC class II-/- DC that had been stimulated with mbCD40L or left untreated. Tumor challenge occurred 7 days after DC immunization. Mean tumor size ± SE and the numbers of animals per group that developed tumors are shown. B, Experimental set-up was as described in A. Data from three separate experiments are combined, and the mean tumor size ± SE on day 25 after tumor challenge is shown. All control animals developed tumors. The percentages of animals that developed tumors in the other groups were: 67%, MHC class II+/+ DC; 100%, MHC class II-/- DC; 60%, CD40L-stimulated MHC class II-/- DC. C, Experimental set-up was as described in B, except that tumor recipients did not receive adoptive transfer of TCR transgenic T cells. All control animals developed tumors. The percentages of animals that developed tumors in the other groups were: 70%, MHC class II+/+ DC; 93%, MHC class II-/- DC; 80%, CD40L-stimulated MHC class II-/- DC.

	Discussion

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The function of CD4⁺ T cells in the generation of CTL responses has been viewed as a provision of cytokines to CD8⁺ T cells (1, 2). However, later studies have suggested that T cell help is more likely mediated via activation of the APC by CD4⁺ T cells (19). Indeed, expression of CD40L on CD4⁺ T cells recognizing cognate Ag presented on DC, and interaction of this ligand with CD40 on DC, was shown to mediate an activating signal that renders DC capable of activating CD8⁺ T cells (7, 8, 9). In agreement with those studies, we show here that stimulating MHC class II^-/- DC with mbCD40L improved the capacity of these cells to induce antitumor immune responses. In fact, the degree of tumor protection induced by immunization with mbCD40L-stimulated, MHC class II^-/- DC was similar to that observed following immunization with MHC class II^+/+ DC. Therefore, the requirement for CD4⁺ T cell help in the initiation of an antitumor immune response can be circumvented by direct ligation of CD40 on DC.

Ligation of CD40 on DC causes an up-regulation of MHC class II molecules and of the costimulatory molecules CD80 and CD86. Others have reported that CD40 ligation induces up-regulation of the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) and increased production of IL-12, TNF-, and IL-1ß (reviewed in 20). It is not clear which, or indeed if any, of these responses define the help function induced by CD40 signaling, although it has been reported that exogenous IL-12 can partially bypass the requirement for CD40 signaling in some antitumor immune responses (21). Alternatively, the restoration of MHC class II^-/- DC function via signaling through CD40 may be explained by effects of CD4⁺ T cell help on the survival of DC in vivo, in addition to their enhanced immunostimulatory capacity. We are currently undertaking experiments to investigate this possibility further.

In repeated experiments, we observed that some degree of CD8⁺ T cell activation and antitumor immunity was induced in mice immunized with MHC class II^-/- DC. This was not due to cross-presentation of tumor Ag on endogenous MHC class II^+/+ DC, as a similar degree of CD8⁺ T cell activation was also observed in MHC class II^-/- mice immunized with Ag-loaded MHC class II^-/- DC (22). In contrast, studies using mice depleted of CD4⁺ T cells before DC immunization and subsequent tumor challenge indicated that antitumor immunity was completely dependent upon the presence of CD4⁺ T cells (23, 24). These discrepancies may be due to differences in the experimental protocols used (MHC class II^-/- DC vs CD4⁺ T cell depletion) or may reflect the different maturation states of the cultured DC used in these experiments. DC cultured from bone marrow precursors in the presence of GM-CSF and IL-4 have been typically referred to as "immature" DC (6). However, we have shown previously that cultured DC possess both the Ag capture capacity characteristic of immature DC and the high stimulatory capacity characteristic of mature DC, perhaps implying an intermediate stage of maturation (18). It is possible, therefore, that even a limited degree of in vitro maturation may provide sufficient activation of DC to permit limited CTL induction in vivo.

We have also compared the degree of tumor protection with or without the transfer of tumor-specific TCR transgenic CD8⁺ T cells before DC immunization. Immunization with MHC class II^-/- DC failed to induce tumor immunity comparable with MHC class II^+/+ DC even in hosts that had been adoptively transferred with TCR transgenic T cells (compare Fig. 5, B and C). This result indicates that a maximal antitumor immune response is primarily dependent upon the availability of T cell help rather than on the size of the CTL precursor pool.

The findings reported here have important implications with regard to the application of DC immunization to the treatment of tumors. Immunization strategies that do not include MHC class II-bound epitopes may fail to attract sufficient CD4⁺ T cell help to allow appropriate DC activation and, in turn, fail to activate CD8⁺ effector cells. In addition, direct in vitro activation of DC with CD40L circumvents the requirement for cognate T cell help, enhancing the immunogenicity of the vaccination protocol. Finally, concerns that terminally mature DC may fail to migrate to regional LNs and thereby fail to initiate antitumor immunity appear to be unwarranted in view of our findings.

	Acknowledgments

 
We thank the personnel of the Wellington Medical School Biomedical Research Unit for animal husbandry and Drs. H. Pircher and H. Bluethmann for the gift of reagents.

	Footnotes

 
¹ This work was supported by a grant from the Cancer Society of New Zealand and an equipment grant from the New Zealand Lottery Board. J.Y. is a visiting scholar supported by the Shanxi Cancer Institute, People’s Republic of China. D.S.R. is supported by Capital Coast Health Limited. F.R. is the recipient of a Wellington Medical Research Foundation Malaghan Senior Fellowship.

² I.F.H. and D.S.R. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Franca Ronchese, Malaghan Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand. E-mail address:

⁴ Abbreviations used in this paper: DC, dendritic cell(s); CD40L, CD40 ligand; LCMV, lymphocytic choriomeningitis virus; LCMV_33–41, LCMV glycoprotein amino acids 33–41; MHC class II^-/-, MHC class II deficient; mbCD40L, membrane-bound CD40L; LN, lymph node.

Received for publication January 19, 1999. Accepted for publication April 8, 1999.

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