The Capacity To Induce Cross-Presentation Dictates the Success of a TLR7 Agonist-Conjugate Vaccine for Eliciting Cellular Immunity This information is current as of October 2, 2021. Jason Z. Oh and Ross M. Kedl J Immunol 2010; 185:4602-4608; Prepublished online 15 September 2010; doi: 10.4049/jimmunol.1001892

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Supplementary http://www.jimmunol.org/content/suppl/2010/09/13/jimmunol.100189 Material 2.DC1 http://www.jimmunol.org/ References This article cites 29 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/185/8/4602.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Capacity To Induce Cross-Presentation Dictates the Success of a TLR7 Agonist-Conjugate Vaccine for Eliciting Cellular Immunity

Jason Z. Oh and Ross M. Kedl

Covalent conjugation of TLR agonists to protein Ags often facilitates the generation of a CD8+ T cell response. However, mechanisms underlying the efficacy of the conjugate over its unconjugated counterpart have been largely uninvestigated. In this study, we show that conjugation of a TLR7 agonist enhances CD8+ T cell responses without affecting Ag persistence and with minimal impact on cellular uptake of the Ag in vivo. Instead, the conjugated form induced a robust accumulation of dendritic cells (DCs) in regional lymph nodes. Perhaps more importantly, cross-presentation in DCs was detected only when the Ag was delivered in the conjugated form with the TLR7 agonist. Collectively, these data represent the first demonstration that a TLR agonist–Ag + conjugate elicits CD8 T cell responses based not on its capacity to induce DC maturation or Ag persistence and uptake, but on the Downloaded from engagement of DC cross-presentation pathways. The Journal of Immunology, 2010, 185: 4602–4608.

oll-like receptor 7 is an intracellular receptor that recog- inflammatory cytokines is not sufficient to bridge the innate and nizes ssRNA molecules and enables the host to sense RNA adaptive immune systems. We previously explored the idea of viruses such as the Influenza virus (1). However, synthetic covalently linking a TLR7a to Ag as a means to mimic the natural

T http://www.jimmunol.org/ compounds agonistic for TLR7 had already been discovered prior recognition of pathogens (6). Using a TLR7a covalently conju- to the identification of RNA as the natural ligand (2). Collectively gated to the HIV-1 gag protein, we and our collaborators demon- known as imidazoquinolines, these compounds structurally re- strated that CD4+ and CD8+ T cell responses can indeed be gen- semble RNA molecules, and similarly to the physiologic ligand, erated in both mouse and nonhuman primates (6, 8). Other studies they induce innate immune activation via TLR7 (2–5). Imidazo- have shown a similar enhancement of the adaptive response when quinolines upregulate costimulatory molecules such as CD80, using Ags conjugated to ligands targeting TLR2, TLR4, or TLR9 CD86, and MHC class II molecules on APCs and induce the (9–16). Thus, the use of chemical conjugation in a vaccine for- localization of dendritic cells (DCs) to the T cell areas of secon- mulation appears to generally enhance the generation of cellular dary lymphoid tissues (5). In addition, rapid induction of inflamma- immune responses. by guest on October 2, 2021 tory mediators, such as TNF-a, IL-12, type I IFNs, and MIP1a However, mechanisms underlying the efficacy of conjugation were reported in mice treated with TLR7 agonists (TLR7a) (2–5). remain poorly defined. Several possibilities exist by which conju- Given the presence of activated APCs and inflammation, which are gation increases the immunogenicity of the Ag and TLR7a. Limited necessary conditions for the priming of T cells, targeting TLRs rep- comparisons between free TLR2 and TLR9 agonist vaccinations resents a rational approach to designing vaccines that can effectively versus their respective conjugates suggested that mechanisms un- induce cross-priming of CD8+ Tcells. derlying the efficacy of the conjugate may be related to increased DC However, despite their ability to activate the innate immune targeting and/or increased Ag uptake (12, 14). Whereas this may be system, TLR agonists, in general, were demonstrated to be rela- a general mechanism by which all TLR agonist–Ag conjugates can tively poor inducers of cellular immunity (6). Immunizations using elicit more potent cellular immunity, it is difficult to imagine how protein Ags mixed with agonists targeting TLRs ranging from agonists targeting TLRs expressed in intracellular compartments TLR2 to TLR9 failed to induce detectable CD8+ T cell responses may mediate this process. Indeed, a TLR9 agonist–Ag conjugate (7), suggesting that simply the induction of APC activation and was reported to be taken up by TLR9-deficient APCs with similar efficiencies as TLR9-sufficient APCs (17), indicating that any in- creased efficacy of Ag uptake is not related to the presence of the Integrated Department of Immunology, University of Colorado, Denver, CO 80206 targeted TLR. Another possibility is that the conjugation of Ag and Received for publication June 11, 2010. Accepted for publication August 12, 2010. adjuvant insures their codelivery into a common endosomal com- This work was supported by National Institues of Health Grants AI068777 and partment, facilitating the efficiency with which the Ag is presented AI170038. J.Z.O. was supported in part by a predoctoral training grant from the Cancer Research Institute. to responding T cells (16, 18). Whereas these and other factors Address correspondence and reprint requests to Dr. Ross M. Kedl, Department of may be necessary, it remains unclear which of these factors truly Immunology, University of Colorado Denver and National Jewish Health, Goodman dictates the success or failure of the vaccination. Comparing these Building K825, Denver, CO 80206 and Dr. Jason Z. Oh at the current address: Emory various parameters between conjugated and free TLR agonist- Vaccine Center of Emory University School of Medicine at Yerkes, 954 Gatewood Road, Atlanta, GA 30329. E-mail addresses: [email protected] (R.M.K.) and based immunizations provides the opportunity to conclusively de- [email protected] (J.Z.O.) termine which of these mechanisms is responsible for facilitating The online version of this article contains supplemental material. vaccine-induced cellular immunity. Abbreviations used in this paper: DC, dendritic cell; dLN, draining lymph node; Lm- In this study, we examined the possibility that conjugation en- OVA, Listeria monocytogenes expressing OVA; LN, lymph node; TLR7a, TLR7 hances the stability of the Ag, resulting in prolonged persistence of agonist. the Ag. In addition, we also considered that conjugation could result Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 in aggregation of the Ag and hypothesized that it would conse- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001892 The Journal of Immunology 4603 quently affect the efficiency of Ag uptake. Using a TLR7a conju- were incubated at the indicated cell numbers with 0.5 3 106 OTI effector gated to fluorescently labeled OVA protein, we found that conju- T cells purified from a 5-d stimulation culture. The magnitude of IFN-g gation did not elongate the t of the Ag, and had a limited impact on production in the effector OTI T cells was used as a measure of the level 1/2 of Ag cross-presentation by DCs. This was quantified as percentage of efficiency of Ag uptake. Instead, conjugation resulted in a dramatic maximum IFN-g response, which was calculated based on the percentage increase in the frequency of DCs accumulating in locally draining of the maximal OTI IFN-g response (achieved by SIINFEKL-pulsed DCs) lymph nodes (LNs). Perhaps more importantly, cross-presentation that is induced by the sorted DCs. was detected in DCs only when the Ag was delivered as a conju- gate to the TLR7a. These results not only clarify the mechanism of Statistical analyses action of this particular adjuvant, but more broadly suggest that the Statistical analyses were performed in Prism (GraphPad, San Diego, CA) timely recruitment of DCs and facilitation of cross-presentation may using Student two-tailed t tests. Asterisk symbols (p) were used to mark the be the key determinants by which any vaccine adjuvant successfully statistical significance of a given difference in sample means and summarize the p value in the following manner: pp , 0.05; ppp , 0.005; pppp , 0.0005. induces cellular immunity. Unless otherwise indicated, shown values of bar graphs are SEM.

Materials and Methods Results Mice and immunizations To understand the underlying mechanism by which conjugation of C57BL/6 and OTI mice were obtained from The Jackson Laboratory (Bar a TLR agonist to Ag serves as a protective vaccine formulation, Harbor, ME) and bred at National Jewish Health. Mice were immunized we conjugated an imidazoquinoline TLR7a to OVA, as previously s.c. in the lower/upper flanks or in the footpad with whole OVA protein described (6, 8), and first examined its ability to establish protective

(OVA; 5–500 mg), or with the TLR7a, 3M012, either in the unconjugated Downloaded from form or as a covalently linked conjugate. OVAwas purchased from Sigma- immune responses. We found that the OVA–TLR7a conjugate eli- Aldrich (St. Louis, MO), and any contaminating endotoxins were eliminated cited a substantial OVA-specific CD8+ T cell response (Fig. 1A), using Triton X-114 and SM-2 Bio-Beads (Bio-Rad, Hercules, CA), as similar to previously published results with the HIV-1 gag protein– previously described (19). The TLR7a, 3M012, was obtained through ma- TLR7a conjugates (6). Moreover, a robust production of IFN-g by terial transfer agreements with 3M Pharmaceuticals (St. Paul, MN). OVA + was covalently conjugated to 3M012, as previously described (6, 8). All CD8 T cells was detected upon a boost injection using the same components injected into mice were tested for LPS contamination using the dose as the primary injection. These data suggest the OVA–TLR7a Limulus amebocyte lysate assay (Lonza, Walkersville, MD) and confirmed conjugate induces functional CD8+ effector T cells and generates http://www.jimmunol.org/ to be free of any detectable levels of LPS. memory CD8+ T cells (Fig. 1B,1C). In contrast, using a 50-fold higher dose of the unconjugated mixture of the TLR7a and OVA Primary and secondary CD8+ T cell responses failed to induce any appreciable primary or secondary response (Fig. OVA-specific CD8+ T cell responses were measured from PBLs of mice 1A,1C), which together highlight the efficiency of conjugation in either 7 d (primary) or 5 d (boost) following injections. Boost injections enhancing cross-priming of CD8+ T cells using minimal amounts of were administered 30–60 d following the primary injection. Cells were then stained with PE-conjugated H-2Kb tetramers loaded with the OVA peptide, whole protein. Most importantly, mice that received only a single SIINFEKL, for 90 min at 37˚C. OVA-specific CD8+ T cells were identified prime and boost injection of the OVA–TLR7a conjugate showed by gating on CD8+ CD42 B2202 events, then gating on tetramer-staining substantial protection against challenge with Lm-OVA. Immuniza- by guest on October 2, 2021 events that were positive for the activation marker CD44. To monitor cy- tion with the OVA–TLR7a conjugate resulted in ∼1000-fold re- + tokine function of effector CD8 T cells, PBLs and spleens of mice were duction in the frequency of bacteria in inoculated mice, demon- harvested 5–6 d postboost injection, pulsed with the SIINFEKL peptide at 1 mg/ml in media containing brefeldin A, and incubated at 37˚C for 4 h. strating a striking ability of the response to control the infection and Cells were fixed and stained for intracellular IFN-g and IL-2 production. lower bacterial burden (Fig. 1D). Given the relatively low frequency of OVA-specific CD8+ T cells generated in the secondary response Protection assay to the OVA–TLR7a conjugate (10–15% of CD8+ T cells) when Mice were immunized with 5 mg OVA–3M012 conjugate in the footpad, as compared with that generated in response to an actual infection described above. Immunized mice were given the same dose of the con- such as vaccinia or lymphocytic choriomeningitis virus (20–60% of jugate as a boost immunization 30–60 d following the primary injection. CD8+ T cells) (20, 21), our results demonstrate the potency by which Approximately 30–60 d following the boost immunization, mice were conjugation of the TLR7a to protein Ags can establish protective challenged i.v. with 2 3 105 CFU Listeria monocytogenes expressing OVA (Lm-OVA). Liver and spleen were removed 3 d after challenge, and the immunity. harvested tissues were mechanically sheared while resuspended in PBS To determine the mechanism by which conjugation enhances the buffer containing Nonidet P-40 (Sigma-Aldrich). They were then cultured efficacy of the TLR7a as a vaccine adjuvant, we examined the on brain heart infusion plates and incubated overnight at 37˚C. The CFU possibility that conjugation increased the t1/2 of the Ag in vivo, was calculated the following day. Lm-OVA was provided by M. Bevan resulting in a depot effect of the Ag. We addressed this hypothesis (University of Washington, Seattle, WA). by utilizing the adoptive transfer of OTI TCR transgenic T cells, Kinetics of Ag uptake, migration, and activation which express a high affinity transgenic TCR specific for the OVA peptide, SIINFEKL. OTI T cells can respond to concentrations as Mice were immunized in the footpad with 5 mg Alexa-488–labeled OVA– low as pico- or femtomolar ranges (22–24), allowing them to serve 3M012 conjugate. At the indicated time points following immunization, DCs were isolated from dLNs using collagenase D and DNase, as pre- as a functional readout for a level of Ag presentation at which the viously described (5). The uptake of Ag by DCs was determined by ex- endogenous OVA-specific T cells may not be responsive. To de- 2 2 amining the frequency of CD11chigh events (pDCa-1 and B220 ) that termine how long Ag persisted in the host in a functionally relevant were positive for Alexa-488 OVA. The magnitude of uptake on a per cell manner, CFSE-labeled OTI T cells were adoptively transferred into basis was determined by measuring the mean fluorescence intensity of Alexa-488 in CD11chigh OVA+ events. mice at 0, 1, 3, or 7 d after antigenic challenge. The loss of CFSE fluorescence was used as an indicator of OTI T cells encountering Cross-presentation assay Ag, and thus an indication that the Ag injected at a particular time prior to the OTI T cell transfer had persisted in vivo. Interestingly, DCs were isolated using either a magnetic bead enrichment method with anti-CD11c (N418) PE mAb and anti-PE microbeads (Miltenyi Biotec, the OVA–TLR7a conjugate showed a pattern of OTI stimulation Auburn, CA) or the MoFlow cell sorter. Purity of the sorted fractions was similar to the unconjugated TLR7a (Fig. 2). Evidence of cellular assessed by flow cytometry and confirmed to be ∼80–90%. Sorted DCs division was detected up to, but not beyond 3 d prior to OTI T cell 4604 TLR7 AGONIST CONJUGATE VACCINE Downloaded from

FIGURE 1. Conjugation of TLR7a to OVA elicits protective CD8+ T cell responses. A, B6 mice were immunized with either a 50 mg mixture of OVA and unconjugated TLR7a or 5 mg OVA–TLR7a conjugate in the footpad and B, given an equal dose of Ag for a boost injection 30–60 d following the primary immunization. Immunized mice were bled from the tail vein 7 d following primary (A) or 5 d following boost immunization (B). Cells were stained with the SIINFEKL-loaded H-2Kb tetramers to measure the frequency of OVA-specific CD8+ T cells. C and D, Mice were given the indicated doses of OVA and 10 mg unconjugated TLR7a or the OVA–TLR7a conjugate. Thirty days later, immunized mice were injected again with the same dose used for the corre- http://www.jimmunol.org/ sponding primary immunization. C, Spleens from the immunized mice were harvested 6 d later, and cell suspensions were prepared and pulsed with the OVA peptide, SIINFEKL, for 4 h. Cells were then stained for intracellular IFN-g. Frequency of IFN-g+ cells shown was gated on live, B2202 CD8+ cells. D, Thirty days following the boost injection, mice were challenged i.v. with 2 3 105 CFU of Lm-OVA per mouse. On day 3, spleens from mice were harvested and cultured on brain heart infusion plates to assess the CFU of Lm-OVA in the spleen as a measure of protection. Dot plots shown (A) are representative of three mice from each sample group. B and C, Graphed values of the frequency of OVA-specific CD8+ T cells are expressed as the mean 6 SEM. Data shown in A–C are representative of four independent experiments, and data shown in D are representative of two independent experiments. Statistical analyses (p) were performed, as described in Materials and Methods. by guest on October 2, 2021 transfer, suggesting that the conjugated TLR7a-Ag does not persist dose of OVA (5 mg) elicited very little response from the trans- in vivo to any greater degree than Ag in the presence of the un- ferred cells (Fig. 3). Whereas the addition of the TLR7a simply conjugated TLR7a. Therefore, the enhanced ability of the OVA– increased the response of the transferred cells to only the highest TLR7a conjugate to generate CD8+ T cell responses is not due to dose of OVA, the transferred cells responded vigorously to a 10- the establishment of an Ag depot in vivo. fold lower dose of the conjugate (Fig. 3). Thus, the differences in In related experiments, however, it was clear that conjugation the effective dose of the Ag required to stimulate the OTI T cells increased the sensitivity of the responding T cells to limiting were exponentially lower for the conjugated than the uncon- amounts of Ag. OTI T cells were adoptively transferred into naive jugated Ag. Collectively, these data indicate that the conjugate recipients, followed by injection of limiting doses of the Ag either facilitates an increased sensitivity to Ag by the responding T cells in combination with, or conjugated to, the TLR7a. The starting (Fig. 3) without the formation of an Ag depot (Fig. 2).

FIGURE 2. Conjugation of the TLR7a to Ag does not increase Ag persistence in vivo. Mice were injected s.c. with 10 mg OVA alone, OVA mixed with the un- conjugated TLR7a, or the OVA–TLR7a conjugate at 0, 1, 3, or 7 d prior to the i.v. transfer of CFSE-labeled TCR transgenic OTI T cells. Locally dLNs were har- vested 3 d following OTI T cell transfer, and the dilution of CFSE was assessed by gating on OTI T cells using SIINFEKL-H-2Kb tetramer+ events. The data shown are representative of two independent experiments. The Journal of Immunology 4605

FIGURE 3. Conjugation increases sensitivity of re- sponding T cells to limiting amounts of Ag. Mice in- jected with CFSE-labeled OTI T cells were challenged with the indicated amounts of the various forms of Ag, as shown. Three days after transfer of OTI T cells, cells from the dLNs were isolated and the OTI T cells were assessed for the loss of CFSE as an indication of the presence of Ag in the host. The histograms shown were gated on live, CD8+, SIINFEKL-Kb tetramer+ events. The data shown are representative of three independent experiments. Downloaded from

We (5, 7, 25) and others (4) have demonstrated the potency with of DCs was observed in mice immunized with the OVA–TLR7a which administration of free TLR7a mediates the activation of conjugate when compared with either OVA alone or the un- DCs in vivo. However, it was still possible that the conjugation of conjugated TLR7a (Fig. 4F). Thus, the overall Ag load appears to the TLR7a enhanced its immunostimulatory properties on the be higher in OVA–TLR7a conjugate-immunized mice, most likely DCs. To test this hypothesis, we compared the level of DC acti- due more to the sizable increase in the number of DCs than to the vation induced by the unconjugated and conjugated forms of the relatively minor increase in the proportion of DCs acquiring Ag http://www.jimmunol.org/ TLR7a. Using the level of MHC class II expression as a measure (Fig. 4F,4G). of DC activation, we found that both forms activated DCs equally Taken together, the data show that whereas the conjugate has at well (Fig. 4A,4B). Nearly 80% of the DCs had significantly up- best a 2-fold enhancement of Ag uptake over time, it enhances both regulated surface expression of I-Ab by 18 h following immuni- DC accumulation (Fig. 4) and sensitivity of T cells to Ag (Fig. 3) zation with either the unconjugated or conjugated forms of the by at least 10-fold. Thus, it was plausible that the increased sen- TLR7a, a dramatic difference from the phenotype observed in sitivity to Ag was simply due to the increased numbers of Ag- non-dLNs. Similar results were obtained for the expression of bearing DCs. Alternatively, the increased sensitivity to Ag may costimulatory markers such as CD86 (data not shown) (5, 7, 25). be due to the capacity of the conjugate to enhance DC Ag pro- by guest on October 2, 2021 Therefore, the data suggest that conjugation status of the TLR7a cessing and cross-presentation on a per cell basis. To test this does not influence the maturation state of the DCs in a fashion that possibility, we developed an assay to quantitate cross-presentation is consistent with the binary outcomes in cross-priming of CD8+ in vivo following immunization with the conjugate and non- T cells (Fig. 1A). conjugate forms of Ag. Mice were immunized s.c. using OVAeither Although the immunostimulatory activities between the two combined with or conjugated to the TLR7a. The DCs from the forms are comparable, a possible explanation for the success of the dLNs were isolated 24 h following immunization and mixed with conjugate over the free TLR7a may be related to differences in their in vitro-stimulated effector OTI cells, and the magnitude of IFN-g capacity to influence overall uptake of the Ag by APCs. To address production by the T cells was measured (Fig. 5A). Because we used this, we labeled OVAwith the fluorochrome, Alexa-Fluor488, with effector T cells in this assay, the presentation of Ag by MHC class I or without further conjugation to the TLR7a, and tracked its uptake is, alone, sufficient for the induction of IFN-g production by the by DCs in immunized mice. Using the labeled OVA, we observed T cells (Fig. 5B). As such, the response of the T cells can be used a rapid uptake by DCs, regardless of whether the TLR7a was as a functional readout of the amount of peptide-MHC complexes conjugated, unconjugated, or absent (Fig. 4C,4D). OVAwas taken on the DC cell surface. up by ∼30–40% of the DCs present in the dLN. Whereas this As expected, DCs purified from mice immunized with the OVA– percentage was enhanced to some degree in mice injected with the TLR7a conjugate exhibited a high level of Ag cross-presentation OVA–TLR7a conjugate, the higher level of uptake was observed (Fig. 5B,5C). In contrast, the unconjugated mixture of TLR7a and only at later time points (Fig. 4D). Furthermore, the overall mean OVA, despite its level of Ag uptake into the DCs (Fig. 4), did not fluorescence intensity values for the fluorochrome-labeled OVA result in cross-presentation. OTI T cells were incubated with a ti- in the conjugated and unconjugated forms did not differ in Ag- tration of increasing cell numbers of DCs (Fig. 5C), and even the bearing DCs at all time points examined (Fig. 4E), suggesting that highest titration of DCs used (4 3 106 cells) did not stimulate any the amount of Ag on a per cell basis is not influenced by the OTI IFN-g production (Fig. 5B,5C). This is significant because conjugation status of the TLR7a. Interestingly, we found this to be we found, at the 24-h time point, relatively similar levels of Ag true not only for the bulk population of DCs, but also for the uptake on a per cell basis between the unconjugated and conju- multiple subsets of DCs present in skin dLNs (Supplemental Fig. gated forms of the Ag (Fig. 4D,4E). In addition, when we lowered 1). Collectively, the data suggest that conjugation had a relatively the amount of conjugate injected to more closely match the minimal impact on either the proportion of DCs that take up the amount of Ag uptake observed using the unconjugated form of the Ag or the duration of Ag uptake by the DCs when compared with Ag, the DCs still cross-presented Ag well above background in the unconjugated form of the Ag. However, we noticed that con- response to the conjugate immunization (Fig. 5D). jugation had a more significant impact on the frequency of DCs in We make two conclusions from these results. First, the observed the dLNs (Fig. 4F). As much as a 10-fold increase in the number failure of DCs to cross-present Ag delivered with the unconjugated 4606 TLR7 AGONIST CONJUGATE VACCINE Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Conjugation does not affect Ag uptake, but enhances DC accumulation in dLNs. A–G, Mice were immunized in the footpad with 5 mg Alexa- Fluor488–labeled OVAwith the unconjugated TLR7a or with the Alexa-Fluor488–OVA-TLR7a conjugate. At 18 h (A, B) or the indicated time points (C–G) following immunization, popliteal LNs draining the foot were harvested, minced, and digested with collagenase/DNase. A, The histograms shown were generated by gating on CD11c+ cells and are representative of four LNs from each treatment group. B, Graphed values of the frequency of DCs expressing high levels of the MHC class II molecule, I-Ab, are expressed as the mean 6 SEM. The frequency of Ag-bearing DCs shown in representative histograms (C) and the percentage of DCs harboring the Ag (D) were calculated based on CD11c+ expression profiles. E, The mean fluorescence intensity values of OVA-AF488 were derived from CD11c+ Alexa-Fluor488+ gated events. The total numbers of Ag-bearing DCs (F) and the entire DC population (G) were calculated based on percentage of CD11c+ or Alexa-Fluor488+ gated events. Statistical analyses were performed, as described in Materials and Methods. The summary of p values, as indicated by (p), denotes statistical significance of differences between the means of the conjugate and OVA + TLR7a treatments. The data shown are representative of three independent experiments.

TLR7a is not due to the Ag uptake falling below some critical Discussion threshold. Second, the DCs from the conjugate-immunized host Mechanisms by which vaccine adjuvants enhance the immuno- have an increased capacity to process and present Ag on a per-cell genicity of Ags have been examined for several decades. However, basis as compared with the DCs from hosts immunized with the a full understanding of the key processes underlying their efficacy unconjugated form of the Ag. The ability to assess DC function on remains unclear and under debate. Early studies have implicated a per-cell basis allows the conclusion that, whereas the conjugate the formation of Ag depots as the underlying cause (reviewed in also augments the accumulation of Ag-bearing DCs in dLNs, the Ref. 26). These findings have led to the proposal that adjuvants critical parameter underlying the efficacy of the conjugated TLR7a enhance the immune response through prolonging the persistence is its enabling of cross-presentation. Thus, the phenotype of DC of Ag in vivo. Additionally, Ag depots are thought to form through cross-presentation highly mirrors the phenotype of the T cell re- aggregation, which may affect the mode and efficiency of uptake sponses generated by the two forms of vaccination; both func- by APCs (27, 28). However, more recent data suggest that the tional outcomes (cross-presentation and T cell expansion) display activation of inflammation via various innate cells and factors is an all-or-none type of a response. The conjugated TLR7a elicited the component dictating the success of an adjuvant (29). To this a robust functional response in both assays, whereas the uncon- end, experimental approaches used to identify alternative vaccine jugated form did not elicit any detectable responses. adjuvants focus now largely on characterizing innate immune The Journal of Immunology 4607 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Conjugation enhances efficiency of cross-presentation by DCs. A, A schematic outline of the method used to assess the level of Ag cross- presentation by DCs ex vivo. Mice were immunized in the footpad with 50 mg OVA–TLR7a conjugate or the unconjugated mixture of OVA and TLR7a. Twenty-four hours following immunization, popliteal LNs were harvested, the cell suspensions from each mouse group were pooled, and the DCs were purified using anti-CD11c magnetic beads. In some experiments, DCs were purified using a cell sorter (MoFlow). Enriched DCs were incubated at 4.0 3 106 cells/well (B) or at the indicated titration of cell numbers (C) with 0.5 3 106 effector OTI T cells. The cells were coincubated for 4 h in the presence of brefeldin A and then stained for intracellular IFN-g. Production of IFN-g by OTI T cells was assessed by gating on expression of the congenic marker CD45.1. The level of IFN-g response by the OTI T cells was expressed as a percentage of the maximal production of IFN-g induced by DCs pulsed with the SIINFEKL peptide. D, Cross-presentation by DCs from mice immunized with either 50 or 25 mg OVA–TLR7a conjugate was assessed as in A–C. The data shown are representative of four independent experiments. receptor signaling pathways and what corresponding impact can- Previous studies by us (6, 8) and others (9–16) have shown that any didate adjuvants targeting these pathways have on the downstream demonstration of these agonists to elicit CD8+ T cell responses adaptive immune system. requires some kind of modification to the way in which the Ag Targeting TLRs through the use of either natural or synthetic and the TLR agonist are delivered. These include coupling the two agonists have been widely explored as a potential alternative components together through chemical conjugation, adsorption by vaccination strategy to induce cellular immunity. This approach has synthetic microparticles, formation of aggregates or Ag depots, or drawn much attention on the basis that TLR stimulation results in the combinatorial use of anti-CD40 mAbs. However, the current robust activation of innate immune cells and the production of assessment of how these methods elicit cellular immune responses proinflammatory cytokines (2–5). However, the assumption that is gravely limited and fails to explain the inability of administering simply inducing these innate immune responses results in cellular the TLR agonist alone to induce such responses. In the current study, adaptive immunity appears to be misplaced. This is particularly we demonstrated the efficacy of codelivering a synthetic TLR7a evidenced by the failure of TLR agonists to elicit CD8+ and CD4+ with a soluble protein Ag through chemical conjugation as a means T cell responses when administered as a mixture with protein Ag to establish cellular immunity. In the process of directly comparing into hosts (6, 7). Therefore, activation of the innate immune sys- this approach with the unconjugated mixture of the TLR7a, we tem by these agonists alone is insufficient to effectively induce identified recruitment of DCs and activation of cross-presentation cellular adaptive immune responses. by DCs as two key determinants governing the success or failure 4608 TLR7 AGONIST CONJUGATE VACCINE of the conjugated and unconjugated forms of the TLR agonist, re- protein to immunostimulatory DNA results in a rapid, long-lasting and potent in- spectively. Our data suggest that, although efficient uptake of Ag by duction of cell-mediated and humoral immunity. Eur. J. Immunol. 30: 1939–1947. 10. Shirota, H., K. Sano, N. Hirasawa, T. Terui, K. Ohuchi, T. Hattori, K. Shirato, APCs and inflammation are necessary, recruiting a sufficient number and G. Tamura. 2001. Novel roles of CpG oligodeoxynucleotides as a leader for of DCs and enabling Ag-bearing DCs to cross-present the acquired the sampling and presentation of CpG-tagged antigen by dendritic cells. Ag are paramount to induce CD8+ T cell responses. J. Immunol. 167: 66–74. 11. Jackson, D. C., Y. F. Lau, T. Le, A. Suhrbier, G. Deliyannis, C. Cheers, C. Smith, DC recruitment and cross-presentation are undoubtedly necessary W. Zeng, and L. E. Brown. 2004. A totally synthetic vaccine of generic structure components mediating CD8+ T cell responses to exogenous Ag. Of that targets Toll-like receptor 2 on dendritic cells and promotes antibody or all parameters critical for vaccine-mediated immunity, the induction cytotoxic T cell responses. Proc. Natl. Acad. Sci. USA 101: 15440–15445. 12. Khan, S., M. S. Bijker, J. J. Weterings, H. J. Tanke, G. J. Adema, T. van Hall, of cross-presentation, in particular, has long been known to hold J. W. Drijfhout, C. J. Melief, H. S. Overkleeft, G. 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