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Adv Dent Res 19:36-41, April, 2006
© 2006 SAGE Publications
Dendritic Cells and HIV Infection: Activating Dendritic Cells to Boost Immunity
Presented at the Fifth World Workshop on Oral Health and Disease in AIDS, Phuket, Thailand, July 6–9, 2004, sponsored by Prince of Songkla University, Thailand, the International Association for Dental Research, the World Health Organization, the NIDCR/National Institutes of Health, USA, and the University of California-San Francisco Oral AIDS Center.
N. Teleshova#,
J. Kenney and
M. Robbiani*
Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021, USA
Correspondence: * corresponding author, mrobbiani{at}popcouncil.org
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Abstract
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Dendritic cells (DCs) are white blood cells that coordinate innate and adaptive immunity. They are distributed within epithelia and mucosal-associated lymphoid tissues, positioned to entrap incoming pathogens or vaccines. Human immunodeficiency virus (HIV) and the non-human primate equivalent (SIV) exploit DCs to amplify infection, underscoring the need to harness strategies that promote presentation of virus by DCs to stimulate potent anti-viral immunity instead of virus transmission. Two main subsets of DCs need to be considered: myeloid (MDC) and plasmacytoid (PDC) subsets. Using the SIV-macaque system to advance oral vaccine research, we examined macaque PDC and MDC biology, identifying ways to activate DCs and boost antiviral immunity. Immunostimulatory oligodeoxyribonucleotides (ISS-ODNs) stimulated PDC/MDC mixtures to up-regulate co-stimulatory molecule expression and to secrete both IFN- and IL-12. Additionally, ISS-ODNs augmented SIV-specific IFN- responses induced by virus-bearing DCs. ISS-ODN-driven DC activation is being pursued to improve oral/nasopharyngeal mucosal vaccines and therapies against HIV.
Key Words: Dendritic cells • immunostimulatory oligodeoxyribonucleotides • SIV • macaque • immunity
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The Macaque-SIV Model
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Congruence between HIV and SIV makes the SIV-macaque system the best animal model for researching the biology of HIV infection (Desrosiers, 1990) and testing preventive vaccine or microbicide strategies needed to impede HIV transmission. We and others have shown that characteristic DCs can be isolated from the tissues and blood of macaques (O’Doherty et al., 1997; Pope et al., 1997; Hu et al., 1998, 1999; Barratt-Boyes et al., 2000; Coates et al., 2003; Teleshova et al., 2004a,b), and that macaque and human DCs drive immunodeficiency virus infection (Frank and Pope, 2002; Teleshova et al., 2003). Paradoxically, however, the normal role of DCs in the immune system is to orchestrate innate and adaptive immunity. One aspect of our research focuses on identifying and testing ways (in macaques) to harness DCs within the oral/nasopharyngeal mucosal-associated lymphoid tissues to enhance preventive and therapeutic vaccine strategies against HIV infection. To achieve this, one must consider the distinctive characteristics of the different DC subsets to which the vaccine should be targeted within these tissues.
The DC system consists of DCs from the myeloid lineage (MDCs) and those from the plasmacytoid lineage (PDCs) (Table ). DCs drive adaptive immunity by secreting numerous cytokines and chemokines and expressing co-stimulatory molecules (e.g., CD40, CD80, CD83, and CD86) on their surfaces to favor the DC-T-cell and DC-B-cell interactions needed for immune stimulation. In addition, viruses induce high levels of IFN- release, especially from PDCs (Siegal et al., 1999; Kadowaki et al., 2000; Fonteneau et al., 2004), to promote DC activation, thereby linking the innate and adaptive responses driving potent anti-viral Th1 immunity (Santini et al., 2000; Biron, 2001).
Further mirroring human DCs (Steinman and Pope, 2002), macaque DCs require stimulation to differentiate into mature, potent immunostimulatory cells capable of inducing strong adaptive T-cell responses (Mehlhop et al., 2002; Frank et al., 2003). This typically involves the DCs up-regulating the expression of co-stimulatory molecules on their surfaces, and the secretion of critical cytokines (e.g., IL-12, IFN-, TNF-). Distinct features of MDCs vs. PDCs highlight their unique roles in coordinating these innate and adaptive events (Table ). In this context, macaque DCs respond comparably to many of the stimuli used to activate human DCs (O’Doherty et al., 1997; Barratt-Boyes et al., 2000; Mehlhop et al., 2002; Coates et al., 2003; Teleshova et al., 2004a,b). Thus, the macaque system provides a relevant model for dissecting the requirements for DC-driven virus infection, how the virus distorts the DC system to its advantage, and how strategies can be designed to bolster DCs to enhance immunity and hamper virus growth.
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Virus Exploitation of Immature Dendritic Cells
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To capitalize most effectively on DC biology to boost immunity to HIV through oral/nasal vaccination, we need to consider the normal DC-HIV interplay that seemingly promotes HIV infection instead of anti-HIV immunity. This is true for at least two reasons. First, boosting DC activity in infected individuals (and overcoming the virus subversion of the DC-driven immune responses) could improve the anti-viral immunity through DCs appropriately presenting the endogenous virus to the immune system. Second, we are investigating the use of a whole, inactivated virus as the vaccine that, other than being non-infectious, mimics live virus in how it interacts with target cells, and needs to be properly targeted to the DCs’ antigen-processing machinery (below).
Complementing extensive human DC-HIV research, several pieces of evidence point to a role for DCs in mucosal transmission of SIV infection, and show how macaque DC-T cell mixtures (reflecting the cellular environments where virus replicates in vivo) promote virus replication in vitro (Frank and Pope, 2002; Teleshova et al., 2003). Even DCs within the mucosal-associated lymphoid tissues of the palatine and pharyngeal tonsils have been shown to be involved in driving virus replication in infected individuals (Frankel et al., 1996, 1997; Hu et al., 1999). Utilizing model monocyte-derived DCs (moDCs), we demonstrated that both immature and mature DCs readily capture large amounts of virus, trafficking virus into distinct locations within immature vs. mature DCs (Frank et al., 2002, 2003; Turville et al., 2004). Despite the dissimilar intracellular localization of the virus within DCs at different stages of maturation, more recent studies visualized the rapid movement of virus from both immature and mature DCs to CD4+ T-cells upon DC-T-cell contact (McDonald et al., 2003; Turville et al., 2004). Some of the earliest in vitro studies on DC-virus biology demonstrated that the DC-T-cell milieu is a site for massive virus amplification (Cameron et al., 1992), even in the absence of exogenous T-cell activation (Pope et al., 1994, 1995, 1997). More recent research with blood-derived MDCs and PDCs (like those found in the tonsillar tissues) demonstrated the susceptibility of these DCs to infection with HIV, and that virus was preferentially transmitted to antigen-specific T-cells (Lore et al., 2005; Smed-Sorensen et al., 2005).
DCs can transmit infection to (resting or activated) T-cells via at least two independent pathways. One involves the direct transfer of internalized virus from the DCs (immature and mature DCs), and the other route requires synthesis of new virus progeny by the infected immature DCs that are then transmitted to the T-cells (Turville et al., 2004). Extremely small amounts of infected immature DCs (undetectable by sensitive quantitative PCR methods) are sufficient to amplify infection upon coming into contact with CD4 T-cells (Turville and Robbiani, unpublished observations). It is also possible that mature DCs can amplify virus (at least to low levels) following signals provided through DC-T-cell interactions (Granelli-Piperno et al., 1999), and then transfer this to the CD4+ T-cells for more extensive propagation.
Additionally, we have shown how a chemically inactivated, non-infectious form of virus (AT-2 SIV/HIV) interacts authentically with human and macaque DCs, and is transmitted from DCs to T-cells, just like live virus (Frank et al., 2002; Turville et al., 2004). This allows us to study early events of DC-virus interplay (independent of infection), as well as to examine AT-2 SIV as a potential vaccine. Specifically, aldrithiol-2 (AT-2) treatment of SIV/HIV renders the viruses non-infectious by covalently modifying the free thiol groups on internal structural proteins, including the cysteines of the zinc finger motifs of the nucleocapsid protein (Gorelick et al., 1999). Importantly, this treatment preserves the conformational and structural integrity of the surface envelope proteins, where cysteine residues are disulphide-bonded (Arthur et al., 1998; Rossio et al., 1998), allowing for genuine cell-virus interplay. From the vaccine perspective, the AT-2-treated virus provides a wealth of antigenic determinants (like live virus) that should induce broad immunity with no risk of infection, and is being tested in the macaque system for its ability to stimulate immunity and protect against or control an infectious virus challenge.
Using AT-2 SIV in the macaque system (Mehlhop et al., 2002; Frank et al., 2003) and AT-2 HIV with human cells (Buseyne et al., 2001; Larsson et al., 2002), investigators have showed moDCs to present antigenic determinants of processed AT-2 viruses and stimulate virus-specific T-cells. In vitro presentation of AT-2 HIV stimulated MHC Class I-restricted CD8+ T-cells, and this required the presence of fusogenic envelope on the virus (Buseyne et al., 2001). This suggests that the exogenously derived virus had to access the cytoplasm by fusing with the cell membrane (from outside the cell or from within an intracellular compartment). Furthermore, using primary SIV-specific T-cells from healthy, infected macaques, we showed that, while mature DCs loaded with AT-2 SIV stimulated both CD4+ and CD8+ T-cell responses, the immature DCs predominantly induced CD4+ T-cells (Frank et al., 2003). Moreover, an AT-2 virus-loaded mature moDC immune therapy reduced the plasma virus loads in both SIV-infected macaques (Lu et al., 2002) and chronic HIV-infected people (Lu et al., 2004). A recent report on intravenously administered SIV peptide-loaded TNF--matured moDCs demonstrated the induction of SIV-specific IFN- T-cell as well as low-level Ab responses in the blood of naïve macaques that correlated with lower viral loads after intravenous challenge with SHIV 89.6P (compared with the control non-immunized group) (Nehete et al., 2005). Analysis of recent data also indicates that subcutaneously injected AT-2 SIV-loaded mature moDCs can prime SIV-specific IFN- and Ab responses in naïve macaques, and that 2 of the 4 immunized animals remained uninfected following rectal challenge with SIVmac239 (MR, unpublished observations). Analysis of these data, together, underscores how mature DCs are able to induce more effective responses involving CD4+ and CD8+ T-cells, as well as Ab responses, that would likely afford better protection against HIV infection than just the CD4+ T-cell responses elicited by virus-bearing immature DCs.
Adding to this more mediocre response induced by the immature DCs, HIV-specific CD4+ T-cells have been shown to exhibit heightened sensitivity to HIV infection (Douek et al., 2002). Therefore, by principally activating CD4+ T-cells during the earliest stages of infection, virus-bearing immature DCs in healthy mucosal tissues would actually encourage infection (transmitting internalized and newly produced virions) in the face of sub-optimal immunity, before the broader CD4+ and CD8+ T-cell responses would be induced.
This is largely driven by the fact that HIV does not activate DCs like other pathogens, subverting the induction of potent immunity (Table). Myeloid-derived DC responses triggered by HIV do not favor strong Th1 immunity unless other stimuli are provided (Frank and Pope, 2002; Messmer et al., 2002; Frank et al., 2003; Teleshova et al., 2003; Granelli-Piperno et al., 2004). Specifically, virus provokes responses in DCs that encourage (i) recruitment of more leukocytes (virus targets) to the initial focus of infection, (ii) DC-leukocyte contact, and (iii) sub-optimal or improper immunostimulatory capacities. Together, these attributes limit the induction of Th1 immune responses while promoting infection. In addition, PDCs secrete type I IFNs, but only a little IL-12, in response to HIV (Soumelis et al., 2002; Fonteneau et al., 2004) or SIV (Teleshova et al., 2004b) (below), implying another route via which immunodeficiency viruses subvert full activation of the DC system. In fact, MDC and PDC numbers may be reduced and their functionality impaired as HIV infection advances (Donaghy et al., 2001; Soumelis et al., 2001), even though MDC and PDC function appeared to be intact immediately following in vitro infection (Lore et al., 2005; Smed-Sorensen et al., 2005). As a consequence, by targeting immature DCs in the epithelia (and in circulation), immunodeficiency viruses avoid inducing effective anti-viral responses, as well as possibly down-modulating responsiveness to other pathogens as disease progresses. This reinforces the importance of identifying effective strategies that will activate DCs to mount strong innate and adaptive responses to prevent the onset of or control established HIV infection.
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Boosting Dendritic Cell Function to Enhance Immunity
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In striving to explore strategies to exploit DCs within the oral/nasopharyngeal mucosal tissues, to improve the immunogenicity of mucosally applied vaccines against HIV, we performed the first detailed study on the ability of a recently described type of immunostimulatory oligodeoxy-nucleotide (ISS-ODN) to stimulate macaque DCs (Teleshova et al., 2004b). ODNs containing unmethylated CpG motifs (ISS-ODNs) activate innate and adaptive immune mechanisms (Krieg, 2002), typically signaling through Toll-like receptor 9 (TLR9) (Hemmi et al., 2000; Bauer et al., 2001). Expressing TLR9, PDCs (not MDCs) and B-cells exhibit differential responsiveness to different CpG-containing DNAs (Verthelyi and Zeuner, 2003) (Table). Type A (CpG-A) ISS-ODNs (also known as D ODNs) typically stimulate IFN- by PDCs, but induce limited DC maturation and do not directly stimulate B-cells. In contrast, the type B (CpG-B) ISS-ODNs (also called K ODNs) directly activate B-cells and up-regulate CD80/CD86 on PDCs, but only low-level IFN- secretion. Recently described type C or CpG-C ISS-ODNs activate human PDCs (sustained IFN- release) and B-cells (Hartmann et al., 2003; Marshall et al., 2003), but it is not yet clear if TLR9 is involved (Verthelyi and Zeuner, 2003). Since IFN- drives DC activation, favoring the induction of Th1 immunity (Santini et al., 2000; Biron, 2001), we rationalized that CpG-C ISS-ODN activation represents a promising approach to activate the DC (PDCs directly and MDCs indirectly) and B-cell systems to augment oral/nasal mucosal vaccine efficacy.
Before pursuing the use of CpG-C ISS-ODNs in vivo, it was essential that we define macaque DC biology in the naïve and healthy, infected animals used to monitor antigen presentation by DCs in vitro, and determine whether CpG-C ISS-ODN exposure augments the presentation of AT-2 SIV by macaque DCs to boost SIV-specific responses. The DCs circulating in the peripheral blood were used as an accessible source of cells to model those within the mucosal-associated lymphoid tissues. These cells would encounter vaccines applied to the oral/nasopharyngeal mucosae and likely be central in the initiation of innate and adaptive antigen-specific immunity. The findings from this study, presented at the Fifth International Workshop on Oral Health & Disease in AIDS, were recently published (Teleshova et al., 2004b) and are outlined below with final discussion on the implications of CpG-C ISS-ODN activation of mucosal DCs (and B-cells) for improving oral/nasopharyngeal mucosal vaccine modalities.
Circulating DC subsets in naïve and infected macaques
In agreement with two earlier reports in naïve macaques (Pichyangkul et al., 2001; Coates et al., 2003), we confirmed the presence of the classic PDC and MDC subsets within macaque blood through four-color flow cytometric analyses. DCs were defined within a population of cells lacking the expression of standard lineage (Lin) markers (found on T-cells, B-cells, monocytes, and NK cells, but typically not on DCs), but expressing high levels of the MHC class II molecule HLA-DR (Lin-HLA-DR+). Within the Lin-HLA-DR+ subset (representing 3–4% of the peripheral blood leukocytes), characteristic CD11c+CD123– MDCs and CD11c–CD123+ PDCs were identified in the blood of naïve and healthy, chronically infected macaques. As in humans, MDCs are the larger population, with MDCs representing 35–60% and PDCs 7–16% of the Lin–HLA-DR+ cells in macaque blood.
Due to the low frequency of DCs in the peripheral blood cell suspensions, we adapted strategies to enrich the DCs by depleting the cells expressing lineage markers. This afforded ~ 10-fold enrichment of the DC-containing Lin–HLA-DR+ fraction within the lineage-depleted cells, to 33–48%. B-cells were the main contaminant remaining in the lineage-depleted fractions. As in the total peripheral blood cell suspensions, the CD11c+CD123– MDCs made up the major proportion of the cells (58–82%), with the CD11c– CD123+ PDCs a small subset (2–16%) of the lineage-depleted cells. It is not possible to obtain sufficient numbers of purified PDC and MDC subsets from blood volumes attainable from macaques to study the cells independently. However, the use of these DC-enriched populations allowed us to move ahead and monitor MDC and PDC biology more closely. Moreover, communication between the two DC subsets is likely integral to DC biology (Fonteneau et al., 2004), and reflects what would be occurring in the lymphoid tissues in vivo.
CpG-C ISS-ODN vs. AT-2 SIV activation of macaque DCs
Four-color flow cytometric analyses verified that the MDCs and PDCs, isolated fresh from the blood of naïve and infected macaques, express low levels of the co-stimulatory molecules CD80 and CD86, typical for immature DCs. Simply culturing the DC-enriched Lin– cell mixtures overnight at 37°C induced at least low-level activation of the DCs. This was evident as the increased surface expression of the co-stimulatory molecules CD80 and CD86, as well as release of IL-12 into the culture supernatants within 24 hrs of culture.
To explore whether we could amplify DC activation by driving IFN- release coincident with more robust elevations in IL-12 release, along with greater CD80 and CD86 expression, we initially compared previously studied and well-characterized stimuli, CD40L and CpG-B ISS-ODN. These stimuli both further increased IL-12 secretion by the Lin– DC-enriched cultures. However, no IFN- release was induced by the CpG-B ISS-ODN stimulation unless IL-3 was added to the cultures. Although the presence of IL-3 did not appear to affect macaque PDC numbers in the cultures, it likely signaled them via CD123 (the IL-3 receptor) to exhibit more potent functionality. Analysis of the membrane phenotypes confirmed that CpB-B ISS-ODN induced increased expression of CD80 and CD86 on the CD123+ PDCs, with some bystander activation of the CD11c+ MDC-containing fraction (above that seen in the control cultures). These responses were similar in uninfected and infected animals, comparable with those seen in humans, although higher IFN- production by CpG-B ISS-ODN-stimulated human PDCs was detected (Marshall et al., 2003).
In light of the low IFN- induced by CpG-B ISS-ODN stimulation of macaque DCs, and knowing that the CpG-C ISS-ODNs stimulated stronger IFN- responses than did CpG-B ISS-ODNs in human cells (Marshall et al., 2003), we examined the responsiveness of macaque PDCs to the CpG-C ISS-ODN, C274. In blood-derived Lin– cells from naïve and infected animals, both CD80 and CD86 expression was significantly up-regulated on PDCs after C274 exposure, with low-level bystander activation of the MDCs (Fig. 1). Furthermore, relative to the control ODN (1040), C274 consistently induced IL-12 as well as IFN- secretion by macaque PDC/MDC mixtures (Fig. 2). Unlike the CpG-B ISS-ODN-stimulated responses, CpG-C ISS-ODN-induced IFN- release did not require, and was not augmented by, the inclusion of IL-3. C274 even induced IFN- secretion from peripheral blood cell cultures where the DCs were not enriched and PDC numbers were extremely low (not seen with the CpG-B ISS-ODN; not shown). This could be explained by the recent observation that C274 induces IL-3 production by blood and lymphoid suspensions (Teleshova et al. 2005; and unpublished data), predominantly by B-cells (Teleshova et al., 2006). This highlights the superior capacity of CpG-C ISS-ODNs to stimulate macaque PDC activation (potentially through direct and indirect effects), just as was described for human PDCs (Hartmann et al., 2003; Marshall et al., 2003). In addition, the bystander MDC activation, combined with the direct PDC activation triggered by the CpG-C ISS-ODN, should provide an effective way to boost the immunostimulatory functions of this DC milieu.
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Fig. 1 - Membrane phenotypic changes induced by CpG-C ISS-ODN vs. AT-2 SIV. Lineage-depleted cells were cultured with 5 µg/mL of the CpG-C ISS-ODN C274 vs. the control ODN 1040 or in medium (Med) vs. 300 ng gag/mL AT-2 SIV E11S (SIV) overnight. The cells were then monitored by four-color flow cytometry for the measurement of CD80 expression by the Lin–HLA-DR+ DC-containing fraction (DC), as well as by the MDC-containing Lin-HLA-DR+CD123– subset (CD123–) and Lin–HLA-DR+CD123+ PDCs (CD123+). The mean values (from three to nine donors) of CD80 expression (mean fluorescence intensity, MFI) by each population cultured under the indicated conditions are shown.
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Since we were using AT-2 SIV as the antigen, and AT-2 HIV has been shown to activate human PDCs (Fonteneau et al., 2004), it was important to evaluate the impact of AT-2 SIV on macaque DC biology. Culture of macaque MDC/PDC mixtures with AT-2 SIV resulted in PDC activation, as evidenced by increased CD80 and CD86 expression (Fig. 1; and not shown) as well as by the release of considerable amounts of IFN- (Fig. 2A; typically greater than that induced by C274). Notably, the bystander activation of the MDC-containing fraction was less apparent upon exposure to AT-2 SIV, compared with that observed in CpG-C or CpG-B ISS-ODN-stimulated mixtures (Fig. 1; and not shown). Furthermore, compared with the C274-induced IL-12 secretion (above the control ODN-cultured cells), AT-2 SIV consistently stimulated limited IL-12 release over that produced by the DC-enriched Lin- cells cultured in medium (Fig. 2B). Thus, despite the membrane phenotypic changes primarily in the PDC subset and increased IFN- release in response to AT-2 SIV, the limited activation of the MDC fraction and poor IL-12 production reflected sub-optimal activation of the total DC population that would need to be boosted to induce the most potent responses. As noted earlier, this underscores one of the mechanisms via which SIV and HIV can avoid effective immune activation, and the fact that exogenous DC stimuli are needed to facilitate effective DC activation for the induction of robust anti-viral immunity.
Increased IFN- triggered by CpG-C ISS-ODNs correlates with increased SIV-specific IFN- release
Our earlier studies revealed that AT-2 SIV activated both CD4+ and CD8+ T-cells in peripheral blood cells isolated from healthy, infected macaques (Frank et al., 2003). Depletion of HLA-DR+ cells completely abrogated theses responses, whereas removal of CD14+ cells only partially affected the responses, and CD20+ B-cell removal had no effect. This suggested that HLA-DR+ DCs and, to some extent, CD14+ monocytes were involved in the presentation of the AT-2 SIV-derived antigens in the peripheral blood mixed-leukocyte cultures. From our moDC studies, we know that mature DCs favor the activation of CD4+ and CD8+ T-cell responses (Frank et al., 2003). We also now know that overnight culture as well as exposure to AT-2 SIV at least partially matures the DCs within peripheral blood cell suspensions (above). Therefore, it is likely that the matured DCs within the peripheral blood cultures were largely responsible for the AT-2 SIV-induced CD4+ and CD8+ T-cell responses.
To verify this, we documented that AT-2 SIV-loaded Lin- cells enriched for DCs stimulated IFN- release from autologous T-cells (from healthy, infected animals containing SIV-primed T-cells). This reinforced that macaque DCs within the Lin– cells could capture the virus and process (at least some of) the viral determinants for presentation to the SIV-specific T-cells. This is supported by the recent findings that human PDCs and MDCs can capture (and become infected by) HIV (Lore et al., 2005; Smed-Sorensen et al., 2005). IFN- release was not detected when cells from naïve (non-SIV-primed) animals were used. When the CpG-C ISS-ODN C274 was added to these cultures for better activation of the DCs, the SIV-specific IFN- responses were increased (augmenting the DC activation already induced through in vitro culture and exposure to the AT-2 SIV). Closer examination of the ability of C274 to enhance these responses was achieved by the addition of titrated doses of AT-2 SIV to cell suspensions enriched for DCs and T-cells (CD14– CD20– cells) along with C274 (vs. the control ODN). In these experiments, we confirmed that AT-2 SIV and C274 together enhanced the SIV-specific IFN- released, and this was most apparent at the lower AT-2 SIV doses (Fig. 3, left Y axis, squares). Since the in vitro culture combined with the (high dose) AT-2 SIV activated the DCs (above), the CpG-C ISS-ODN effect was not as dramatic at the highest AT-2 SIV concentration.
Of note, the enhanced IFN- production induced in the presence of C274 and AT-2 SIV coincided with elevated IFN- secretion in these cultures (Fig. 3, right Y axis, triangles). IFN- secretion was not detected (at this cell number) with either stimulus alone. The actual pg/mL levels of IFN- were lower in the presence of less AT-2 SIV. However, when the CpG-C ISS-ODN was added to the low dose of AT-2 SIV, the greater IFN- released in the presence of CpG-C ISS-ODN C274, compared with that secreted by the control ODN-stimulated cells, was still evident. Consequently, the CpG-C ISS-ODN-induced DC activation—manifest as increased expression of CD80 and CD86, elevated IL-12, and IFN- release—translated to enhanced SIV-specific IFN- production being induced by the AT-2 SIV-bearing DCs.
CpG-C ISS-ODN functions in vivo and activates macaque B-cells as well as DCs
Extending our research on blood-derived DCs, we have monitored DC and B-cell responses to CpG-C ISS-ODN C274 in lymphoid cell suspensions, as well as extensively analyzed blood B-cell responses to C274 for comparison (our unpublished observations). Evaluating blood from naïve and healthy, infected animals, we have found that CpG-C ISS-ODN C274 readily activates macaque B-cells in vitro. C274-activated macaque B-cells proliferate, up-regulate co-stimulatory molecule expression, and secrete various cytokines and chemokines (Teleshova et al., 2006). C274-induced B-cell activation is typically more robust than that induced by CD40L or the CpG-B ISS-ODN.
Importantly, C274 also stimulates macaque DCs and B-cells isolated from lymphoid tissues and functions in vivo (unpublished data). To investigate this in a controlled manner, we utilized intranodal injection of C274 into superficial lymph nodes (vs. C661 injected into the contralateral lymph nodes within the same animal), to model ODNs accessing the mucosal-associated lymphoid tissues following topical application of a vaccine plus ODN adjuvant. This work revealed that in vivo C274 exposure heightens the responsiveness of lymphoid DCs and B-cells to subsequent in vitro stimulation. This is manifested by elevated CD80 and CD86 expression by both cell types, as well as by increased IFN-, IL-6, IL-12, and chemokine (e.g., RANTES, MIP-1, MCP-1) release by the stimulated lymph node cell cultures. Complementing our observations with blood DCs and B-cells, these data provide proof for the in vivo activation of lymphoid B-cells and DCs by CpG-C ISS-ODN C274 in naïve and healthy infected macaques.
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Implications for Advancement of Mucosal Vaccines
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The CpG-C ISS-ODN C274 effectively activates macaque DCs and B-cells in vitro and in vivo. This is true for cells circulating in the blood and those located within the lymphoid tissues, like those associated with the oral and nasal mucosae. The membrane-phenotypic modification, combined with the release of IFN-, IL-12, and various chemokines triggered by C274, would favor the induction of the more potent Th1 immunity likely needed to combat HIV. In addition, we documented that circulating DCs can capture and present AT-2 SIV to activate SIV-specific T-cells, and that, coincident with elevated IFN- secretion, C274 further enhanced the SIV-specific IFN- responses stimulated by this antigen.
These data are especially encouraging for the use of CpG-C ISS-ODNs to boost the immunogenicity of a vaccine applied to the mucosal-associated lymphoid tissues. This is particularly the case for the inactivated virus vaccine, where the AT-2 SIV would cross the mucosal barriers just like live virus, and act in concert with the CpG-C ISS-ODN to enhance innate and adaptive immune functions. Analysis of data from a preliminary (proof-of-principle) pilot study in macaques suggests that application of ISS-ODN and AT-2 SIV to the mucosal surfaces of the palatine tonsils induced stronger SIV-specific IFN- responses, compared with those seen when the control ODN and AT-2 SIV were applied (NT and MR, unpublished observations). More comprehensive in vivo macaque studies are required to test whether vaccines applied to the oral/nasopharyngeal mucosae can be improved via the triggering of the immune system through modalities like CpG-C ISS-ODNs, to elicit systemic and mucosal immune responses that will ultimately protect against infectious challenge and control established infection to limit virus spread.
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Acknowledgments
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The authors thank Jennifer Jones for additional technical assistance, as well as Jeffrey D. Lifson for providing the AT-2 SIV, Agegnehu Gettie, Jason Dufor, and Rudolf Bohm for assistance with the macaque sampling, and Jason Marshall and Gary Van Nest for providing the ODNs. MR receives support from the Elizabeth Glaser Pediatric AIDS Foundation, as well as from NIH grants R01s AI040877, DE015512, and DE016256, R21s AI060405 and DE016534, P01s AI052048 and HD041752, and U19 AI065413, the USAID Cooperative agreement GPO-A-00-04-00019-00, and the Tulane National Primate Research Center base grant RR00164. MR is an Elizabeth Glaser Scientist. Some of the data in this study have been published previously (Teleshova et al., 2004b).
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Footnotes
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# current affiliation: Division of Infectious Diseases, Mt. Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA 
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Advances in Dental Research, Vol. 19, No. 1,
36-41 (2006)
DOI: 10.1177/154407370601900108
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M.C. Herzberg, A. Weinberg, and S.M. Wahl
(C3) The Oral Epithelial Cell and First Encounters with HIV-1
Advances in Dental Research,
April 1, 2006;
19(1):
158 - 166.
[Abstract]
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Abstract
The Macaque-SIV Model
