TLR-activated dendritic cells enhance the response of aged naïve CD4 T cells via an IL-6 dependent mechanism

Abstract

The most effective immunological adjuvants contain microbial products, such as toll like receptor (TLR) agonists, which bind to conserved pathogen-associated recognition receptors. These activate dendritic cells (DC) to become highly effective antigen-presenting cells (APC). We assessed whether TLR ligand-treated DC can enhance the otherwise defective response of aged naïve CD4 T cells. In vivo administration of CpG, PolyI:C and Pam₃CSK₄ in combination with antigen resulted in the increased expression of costimulatory molecules and MHC class II by dendritic cells (DC), elevated serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that in vitro, pre-activation of DC by TLR ligands makes them more efficient APC for aged naïve CD4 T cells. Following T:DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR pre-activation of both bone marrow derived and ex vivo DC improved responses. IL-6 produced by the activated DC during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APC by TLR ligands.

Introduction

The age-associated decrease in the function of the immune system contributes to the increased susceptibility to infectious diseases, such as influenza, seen in the elderly (1–7). With advanced age, responses to new pathogens and to vaccines containing new antigens are preferentially reduced. In the aged, efficacy of vaccination is limited by reduced antigen-specific B cell expansion and differentiation (8) and the antibodies that are produced are less functional due to reduced class switching and somatic hypermutation (9). In mice, it has been shown that this is due in large part to the age-associated decline in cognate B cell help by aged naïve CD4 T cells, which we have shown are profoundly defective in helping to generate germinal center B cells and high titers of antigen-specific serum antibody (10). The responses of aged naïve CD4 T cells also display well-characterized defects in IL-2 production, expansion, effector generation, and they become poorly responsive memory cells (11–17). Our more recent studies suggest that a limited complement of defects arise initially from intrinsic changes that are already present in the aged CD4 T cell recent thymic emigrant (RTE) populations (14) and that more severe defects develop as the cells themselves age in the periphery (15–17).

Recent work by our lab and others has highlighted the valuable antibody-independent role that fully competent CD4 effectors and memory cells can play during viral infection, both as helpers of the CD8 T cell response (18) and as direct mediators of viral clearance (19, 20). Thus, defects in the aged CD4 T cell response are likely to cause both markedly diminished humoral and cell-mediated responses against infections with either new viruses or altered strains as well as the failure to develop protective immunity.

The in vitro defect in the expansion of aged CD4 T cells and generation of effectors can be overcome by the addition of exogenous IL-2, but not other IL-2Rγc-binding cytokines (11, 14). However, effectors “rescued” by exogenous IL-2 did not become functional memory CD4 T cells (13, 21). Recently, we demonstrated that in the presence of inflammatory cytokines (IL-6, TNFα, IL-1) in vitro or when these cytokines, or a strong adjuvant such as CFA, were included as part of an in vivo immunization regimen, the response of aged CD4 T cells was greatly enhanced (12). Thus, we considered that the pre-treatment of antigen-presenting cells (APC) with adjuvants that could enhance their production of inflammatory cytokines (22) could potentially stimulate stronger naïve T cell responses against new antigens of emerging pathogens or new strains that have mutated to evade current immunity.

Aluminum hydroxide (alum) is the most commonly used adjuvant in human vaccines. However, its application is limited by its stimulation of Th2 responses and thus by its limited ability to enhance protection against intracellular parasites such as TB, HIV and influenza (23, 24). Toll like receptors (TLR) are expressed by many innate immune cells, including dendritic cells (DC) and other APC, endothelial cells, fibroblasts, and lymphocytes. TLR have recently garnered much attention for their role in initiating the innate immune response against infectious pathogens (reviewed in 25). Each of the 10 known TLR recognizes a different “pathogen associated molecular pattern” (PAMP) expressed by bacteria, viruses and fungi. During an infection, triggering of TLR on DC stimulates their migration to regional lymph nodes (LN) via their down-regulation of CCR5 and up-regulation of CCR7 (26). In the LN, the TLR-activated DC promote strong T cell responses via their increased co-stimulatory molecule and MHC expression (27). TLR activation of DC, and of other APC, induces production of a number of inflammatory cytokines such as IL-1, IL-6 and TNFα (28, 29), as well as anti-viral cytokines such as IFNα and β (27), and IL-12 (27, 30), which is critical for Th1 polarization.

The ability of TLR ligands to influence the subsequent adaptive immune response has stimulated great interest in their use as vaccine adjuvants (30). Many current vaccines that contain whole organisms also contain intrinsic microbial ligands for TLR, but in non-replicating vaccines the level of TLR ligand expression may be too low to act as an effective adjuvant. Thus, the level and role of TLR activation associated with current inactivated or subunit vaccines is unclear (27). Pre-clinical studies using TLR ligands such as viral dsRNA, eg, polyinosinic-polycytidylic acid (polyI:C; TLR3 ligand) and bacterial unmethylated CpG DNA (CpG; TLR9 ligand) have shown that the addition of exogenous TLR ligands during priming can enhance the humoral and/or cellular response to HIV-1 (31), hepatitis B (32), leishmania major (33), influenza (34) and tumor model (35, 36) antigens.

There are conflicting data concerning the effect of age on the responsiveness of APC to stimulation by TLR agonists. Studies with macrophages from age mice have shown decreased TLR gene expression (37), reduced cytokine secretion (37, 38) and intracellular signaling (38) in response to TLR stimulation. Analysis performed on human monocytes revealed depressed cytokine secretion in the aged in response to Pam₃CSK₄, which signals through TLR1/2 (39, 40), and a more general defect in the upregulation of CD80 expression in response to a broad spectrum of TLR ligands (40). In contrast to these studies, data from a recent mouse study (41) suggests that activation and maturation of aged DC in response to a broad range of TLR agonists is intact. Most important in this study was the finding that both young and aged DC effectively primed a T cell response (41). In addition, a study in humans revealed that aged DC undergo normal phenotypic activation following LPS stimulation, however, these cells secreted elevated amounts of inflammatory cytokines (42). Maue et al (43) recently demonstrated that including a TLR ligand in an in vivo immunization regimen results in an increased aged T cell-dependent B cell response. However, because of the wide range of cells that express TLR in vivo, the mechanism by which this TLR agonist is enhancing the helper cell response is unknown. Thus, there remains relatively little data examining the extent to which TLR ligands acting on APC can act as adjuvants to improve the response of aged naïve CD4 T cells, and the mechanisms by which TLR stimulation of APC can affect the outcome of aged T cell stimulation remain unclear. Moreover, the broad range of cells expressing TLR means that systemic administration of TLR ligands would have pleiotropic effects on many targets, likely including some that could be deleterious which would limit their use, especially in the elderly. If targeted activation of APC is sufficient to overcome aged naïve CD4 T cell defects, then new strategies could be developed to exploit this ability, thereby avoiding many potential problems of systemic administration.

Here, we find that the TLR agonists CpG, PolyI:C and Pam₃CSK₄ (TLR2/1 ligand) are effective in activating the DC of aged and young mice in vitro as well as in vivo, and we show that the addition of CpG to the immunization regimen enhances the in vivo antigen-specific response of aged as well as young naïve CD4 T cells. To define the mechanisms responsible for this adjuvant activity, we have developed an in vitro model system to assess the hypothesis that direct stimulation of DC by TLR ligands enhances their APC function and subsequent ability to prime aged naïve CD4 T cells, and that this results in a reversal of key aspects of age-associated unresponsiveness. Our results indicate that TLR pre-activated DC used as APC induce a substantial increase in the antigen-specific expansion of aged naïve CD4 T cells, resulting from a combination of increased proliferation and enhanced survival with greater expression of Bcl-2 by activated CD4 T cells. The enhanced response depends upon IL-6 whose production by DC during cognate interaction with the T cell is greatly increased. These studies define key components responsible for enhancing defective responses of aged naïve CD4 T cells and illustrate the potential of targeted vaccine approaches in the aged that take advantage of the natural adjuvant properties of TLR ligands.

Materials and Methods

Mice

HNT H-2^d/d TCR Tg mice express a Vβ8.3 (Vα unknown) transgene that recognizes the 126–138 fragment of the PR8 influenza hemagglutinin (HA) molecule in the context of I-A^d (44). HNT TCR Tg, C57BL/6 H-2^b/b (B6) and BALB/c H-2^d/d (BALB/c) were bred at the Trudeau Institute Animal Facility. All mice were fed sterile standard diet ad libitum and housed in isolator cages under specific pathogen free conditions. Mice referred to as young were 6–10 wk of age; aged B6 and BALB/c mice were 17–24 mo-old; aged HNT TCR Tg mice were at least 15–17 mo old. Aged mice were inspected for gross pathology and animals exhibiting pathology were excluded from experiments. The Trudeau Institute Institutional Animal Care and Use Committee approved all experimental animal procedures.

Medium and antigens

All cells were grown in complete RPMI 1640 (Invitrogen Life Technologies) containing 2mM L-glutamine, 100 IU penicillin, 100 μg/ml streptomycin (all obtained from Invitrogen Life Technologies), 50 μM 2-ME (Sigma-Aldrich), and 8% FBS (HyClone). OVA peptide 323–339 (OVA_323–339) (ISQAVHAAHAEINEAGR), and HA peptide 126–138 (HA_126–138) (HNTNGVTAACSHE) were synthesized by New England Peptide.

Flow cytometric analysis

For cytometric analysis, cells suspended in PBS supplemented with 2% BSA and 0.1% NaN₃ were incubated with fluorochrome-conjugted Abs for 30 min on ice and in the dark. Cells were either analyzed immediately or fixed in 1% paraformaldehyde. MAbs used for these studies were specific for CD11c, CD11b, CD54, CD80, CD86, CD40, IA^d, H2^b, IA^b, CD4, CD8, Vβ8.3, CD25, CD44, CD62L, CD162, IL-6 and the appropriate irrelevant isotype controls. Flow cytometric data were acquired on a FACSCalibur (BD Biosciences) cytometer using CellQuest (BD Biosciences) software. Analysis of cytometric data was done using FlowJo version 6.1.1 software (Tree Star).

In vivo TLR administration and priming

Young and aged B6 mice were injected subcutaneously in the subscapular region with the indicated TLR ligands [(CpG (60μg), PolyI:C (100μg), Pam₃CSK₄ (50μg)]. At the indicated timepoints serum was harvested and cytokine levels were determined by either ELISA or the Luminex Cytokine Bead Array assay (Millipore) as per manufacturer’s instructions. When indicated, draining lymph nodes (DLN) were harvested, teased apart, digested with collagenase D at 37C for 1 hr and strained, after which cells were prepared for FACS analysis as indicated. For priming experiments, young and aged B6 mice were primed i.p. with 50 μg of OVA_323–339 in PBS, with alum (Pierce) or with alum + 60 μg of CpG. Twenty-three days later spleens and peripheral lymph nodes were harvested from primed animals. CD4 T cells were positively selected using magnetic beads and restimulated with syngeneic spleen cells and relevant peptide in ELISPOT plates coated with anti-IL-2. ELISPOT analysis was performed as previously described (45).

Isolation of young and aged CD4 T cells

CD4 T cells were isolated from HNT TCR Tg mice as previously described (16). Briefly, young and aged CD4 T cells were isolated with anti-CD4 beads according to the manufacturer’s protocol (Miltenyi Biotec) and then stained with Cy-Chrome-anti-CD4, FITC-anti-Vβ8.3, APC-anti-CD44, and PE-anti-CD62L. Naïve CD4 HNT T cells were sorted to be CD4⁺Vβ8.3⁺CD44^loCD62L^hi using a FACS Vantage SE. Purity of all sorted populations was ≥95%. In some experiments sorted cells were labeled with the dye CFSE (Molecular Probes) as previously described (16). When indicated, purified cells were stimulated with plate bound anti-CD3 and anti-CD28 as previously described (46).

Bone marrow-derived DC

Bone marrow-derived dendritic cells (BMDC) were generated as previously described (47), with minor changes. Briefly, bone marrow was flushed from femurs and tibia of either young or aged BALB/c or B6 mice and plated at 5×10⁵ cells/ml in complete RPMI plus 5ng/ml recombinant murine GM-CSF (PeproTech). On days three and five, one half of the culture volume was replaced with fresh complete RPMI + GM-CSF. The non-adherent cells were harvested on day seven and characterized to be CD4⁻, CD8a⁻, CD11b⁺, CD11c⁺, CD54⁺, CD80⁺, CD86⁺, MHC class I/II⁺. Harvested BMDC were routinely enriched using directly conjugated CD11c magnetic beads and a MACS column as per manufacturer’s instructions (Miltenyi Biotec.). When indicated, CD11c-enriched BMDC were incubated for 24 hours with 5ng/ml GM-CSF plus TLR adjuvants, such as CpG 1826 (1μM; type B unmethylated CpG-ODN; IDT), PolyI:C (10μg/ml; Sigma or Invivogen), or Pam₃CSK₄ (300ng/ml; Invivogen). The working concentration of each TLR ligand was based upon preliminary studies in which we determined the amount needed to consistently induce the activation of BMDC as assessed by the upregulation of CD80 and CD86. When indicated, BMDC culture supernatants were collected and analyzed for cytokine production using the Luminex Cytokine Bead Array assay (Millipore) as per manufacturer’s instructions. TLR ligands were reconstituted with endotoxin free PBS. Controls used to assure that the results were not influenced by contamination of the TLR ligands by endotoxin included the use of non-CpG ODN to ensure that BMDC stimulation by the ODN preparations was dependent upon engagement of TLR 9 (Non-CpG ODNs did not activate BMDC, data not shown). Endotoxin levels in Pam₃CSK₄ and PolyI:C were lower than 0.125 EU/ml as measured with the LAL assay (as performed/certified by Invivogen). Finally, the phenotypic activation of BMDC by CpG, PolyI:C, and PAM₃CSK₄ was found to be independent of TLR4 signaling as demonstrated by TLR ligand incubation and phenotypic analysis of BMDC derived from wild-type control versus TLR4KO BMDC (Supplemental Data Figure 1; TLR 4 KO mice were a generous gift from Richard Flavell, Yale University).

Splenic dendritic cells

Splenic dendritic cells were generated as previously described (48), with minor changes. Briefly, spleens were minced and dissociated in Spleen Dissociation Media from Stem Cell Technologies (Vancouver, BC, Canada) as per manufacturer’s instructions. Red blood cells in the suspension were lysed with ACK lysis buffer. The cells were then enriched by negatively selecting for B19 and Thy1.2 by directly conjugating to B19 and Thy1.2 magnetic beads and a MACS column as per manufacturer’s instructions (Miltenyi Biotec). Post enrichment, cells were stained with FITC-anti-Thy1.2, PE-anti-B220, PECy5.5-anti-CD8a, and APC-anti-CD11c. Splenic DC were sorted to be Thy1.2⁻B220⁻CD8a⁻CD11c⁺ using a FACS Vantage SE. Purity of all sorted populations was ≥95%. Sorted splenic DC were incubated overnight PolyI:C (10μg/ml) and CpG 1826 (1μM). The working concentration of each TLR ligand consistently induced the activation of splenic DC as assessed by the upregulation of CD80, CD40 and CD86.

T cell culture with BMDC

Following stimulation with TLR ligands BMDC or splenic DC were washed extensively, and when indicated, labeled with CellTrace^™ Violet (Molecular Probes) as per manufacturer’s instructions. DC were then placed into culture with sorted CD4⁺Vβ8.3⁺CD44^loCD62L^hi HNT TCR Tg cells at a 10:1 T:DC ratio. HNT peptide was added at a final concentration of 5μM. After 2 days an equal volume of complete tissue culture medium was added. For [³H] thymidine incorporation assays, cells were pulsed with [³H] thymidine 18 h before the indicated day of culture and then harvested onto glass fiber filters. CPM were determined by scintillation counter. Culture supernatant levels of cytokine were determined either by a bioassay with NK-3 cells (IL-2) or by Luminex Cytokine Bead Array assay as per manufacturer’s instructions. Cell survival was determined by 7-AAD staining as per manufacturer’s instructions (BD Pharmingen). When indicated, 10μg/ml blocking anti-IL-6, anti-TNFα or irrelevant isotype control antibodies (Trudeau Institute Monoclonal Antibody Core), or the indicated amount of IL-6 recombinant cytokine (R&D Systems) were included in the CD4 T cell:DC culture.

Intracellular staining

Intracellular cytokine staining (ICCS) for Bcl-2 and IL-6 was performed as previously described (49). Briefly, cells were surface stained, washed and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were washed and resuspended in saponin buffer (PBS containing 1% fetal bovine serum, 0.1% NaN₃, and 0.1% saponin) containing anti-mouse Bcl-2 (BD Pharmingen) or anti-mouse IL-6 (BD Pharmingen) or isotype control and incubated for 20–30 min at room temperature. Samples were then washed with PBS and analyzed immediately on a FACSCalibur flow cytometer.

Statistical analyses

Statistical analyses were performed by Prism 4.0 software (GraphPad) using student’s t test. Values of p < 0.05 were considered significant and are indicated by an asterisk(*).

Results

CpG enhances the aged CD4 T cell response in vivo

The use of adjuvants that contain TLR ligands has been proposed as a means to enhance the immune response of elderly individuals who respond poorly to current vaccination strategies (50). It is unclear whether TLR ligand-activation only of APC can enhance the antigen specific aged naïve CD4 T cell response and if so what mechanisms might be involved. To confirm the responsiveness of aged in vivo APC, we tested whether TLR agonists could induce in situ activation of myeloid DC in aged as well as young mice. After an initial test of agonists to TLR2-5, 7 and 9, we chose 3 TLR ligands for these in vivo studies: CpG (TLR9 ligand), PolyI:C (TLR3 ligand), and Pam₃CSK₄ (TLR2/1 ligand). We chose CpG and PolyI:C because both TLR3 and 9 are expressed in endosomes, and are capable of inducing DC to produce inflammatory cytokines and promote the development of Th1 CD4 T cells (30). In contrast, TLR2 is expressed on the extracellular plasma membrane and is capable of promoting either a Th1 or a Th2 type of response (30).

Young and aged B6 mice were injected subcutaneously with either CpG, PolyI:C, or Pam₃CSK₄. Twenty-fours hours later, the draining lymph nodes (DLN) were removed and the cells were stained to assess CD86, CD40, and I-A^b expression on CD11c⁺CD11b⁺ cells, which represent the predominant myeloid DC. As seen in Figure 1A, B CD11c⁺CD11b⁺ cells from young and aged mice treated with TLR agonists expressed markedly higher levels of these co-stimulatory molecules that are known to be key in activating naïve CD4 T cells, compared to animals of the same age that received only PBS. In addition, CD11c⁺CD11b⁻ lymphoid DC from young and aged mice were found to be similarly activated following injection of CpG, PolyI:C, and Pam₃CSK₄ (data not shown). This data confirms, as previously suggested, that these key features of TLR activation of DC are intact in aged mice treated with each ligand (41).

Figure 1.

Immunization with TLR ligands enhances the aged CD4 T cell response. Young (A) and aged (B) B6 mice were injected subcutaneously with either CpG (60μg), PolyI:C (100μg), Pam₃CSK₄ (50μg), or PBS. 24 hours after injection CD11c⁺CD11b⁺ dendritic cells from the draining lymph nodes were analyzed for their expression of CD86, CD40 and I-A^b. Dashed line: isotype control; shaded area: PBS treated; solid line: TLR ligand treated. Data are representative of 2 similar experiments. C, Young and aged B6 mice were immunized intraperitonealy with either OVA_323–339 alone, OVA_323–339 + alum, or OVA_323–339 + alum + 60 μg CpG. 21 days after immunization CD4 T cells enriched from the spleen and peripheral lymph nodes were restimulated with OVA_323–339, and the number of IL-2 producing cells was determined by ELISPOT. Data are representative of 2 similar experiments. * indicates p<0.05 between indicated groups.

We have previously shown that the addition of alum by itself to the immunization regimen is not sufficient to enhance the response of aged CD4 T cells (12). To determine whether or not the addition of a representative TLR agonist, CpG, to the alum/antigen immunization regiment could enhance the response of aged CD4 T cells in vivo, young and aged B6 mice were injected with either OVA_323–339 alone, OVA_323–339 in alum, or OVA_323–339 in alum + CpG. Twenty-one days after priming, CD4 T cells enriched from pooled spleen and lymph nodes were re-stimulated with antigen and the number of IL-2-producing cells was assessed by ELISPOT. We found that the inclusion of alum alone was effective in enhancing the young CD4 T cell response to peptide antigen. However, consistent with our previously published data (12), alum by itself was unable to significantly boost the aged CD4 T cell response. In contrast, the inclusion of CpG in the vaccine formulation led to a significant increase in the number of primed aged CD4 T cells that produced IL-2 upon re-stimulation ex vitro. These data illustrate that aged mice have an intact response to TLR agonists which activate in situ DC to up-regulate co-stimulatory and MHC class II molecules and enhance the aged naïve CD4 T cell response.

This improved CD4 T cell response could be the result of TLR agonist action on DC or on other APC leading to enhanced antigen presentation or inflammatory cytokine production, or it could result from direct stimulation of the CD4 T cells by the TLR ligand (51, 52). Alternatively, inflammatory cytokines from other TLR-agonist-activated cell types, such as macrophages, B cells, neutrophils, and endothelial cells, could contribute to enhancing the aged CD4 T cell response. These possibilities would be difficult to evaluate in vivo, so we developed an in vitro model to isolate the effects of TLR agonists acting directly on APC and to determine mechanisms by which the activated DC might subsequently “rescue” aged responses of naïve aged CD4 T cells.

Antigen presentation by TLR-activated dendritic cells enhances the aged CD4 T cell response in vitro

To determine whether the direct activation of DC by TLR agonists could generate APC capable of overcoming defects in the aged CD4 T cell response to antigen, BMDC were TLR-stimulated, washed, and then used as APC in a cognate T cell response model. We generated “immature” BMDC with GM-CSF and enriched for CD11c⁺ DC that seemed to be the equivalent of immature myeloid DC (53). When these cells were stimulated for 24 hours by either CpG, PolyI:C, or Pam₃CSK₄, they expressed markedly higher levels of CD80, CD86, and CD40 and secreted elevated levels of TNFα, RANTES, KC, IL-6, IL-12p70, and IL-12p40 than unstimulated BMDC (Supplemental Data Figure 2). Of most importance, BMDC derived from aged bone marrow of BALB/c mice were as equally responsive as young BMDC to TLR stimulation. This finding was indicated by the increased expression of CD80, CD86 and CD40 (Figure 2A), as well as by the increased secretion of KC, RANTES, IL-6, IL-12p70, and IL-12p40 by both young and aged BMDC following stimulation by CpG (Figure 2B). The increased production of inflammatory cytokines in the young and aged BMDC cultures following TLR stimulation was mirrored by elevated levels of these cytokines in the serum of young and aged BALB/c mice following the in vivo administration of CpG and PolyI:C, indicating similar effects of TLR-ligand administration in vitro and in vivo (Figure 2C).

Figure 2.

BMDC derived from young and aged BALB/c bone marrow and stimulated with CpG demonstrate increased costimulatory molecule expression and inflammatory cytokine production. Mature CD11c-enriched, GM-CSF derived BMDC from young and aged mice were cultured for 24 hours with CpG after which BMDC expression of CD80, CD86 and CD40 was assessed (A), and levels of cytokines in culture supernatants were measured by cytokine bead array (B). A, Dashed line: isotype control; shaded area: PBS treatment; solid line: TLR treatment. Data are representative of 2 similar experiments. C, Young and aged BALB/c mice received a combined injection of CpG (60μg) and PolyI:C (100μg) and were bled for serum cytokine analysis 4 hrs later. Data are combined from 5–7 animals per group.

Purified naïve (CD44^loCD62L^hi) CD4 T cells from aged HNT TCR transgenic mice that express a TCR specific for influenza hemagluttinin (HA) and recognize A/PR8/34 influenza (54) exhibit a profound dysfunction relative to their young counterparts following encounter of cognate antigen (HA_126–138) presented by B cell blasts in vitro, including diminished IL-2 production and CD25 expression, and reduced expansion (16). These in vitro defects correlate with defects in response to antigen in vivo, which included a diminished capability to produce IL-2 and a failure to provide efficient cognate help to a B cell response (16). In this study, we analyze the in vitro response of purified naïve HNT Tg CD4 T cells from young and aged mice, sorted on the basis of their naïve phenotype to be CD4⁺Vβ8.3⁺CD44^loCD62L^hi. The naïve CD4 T cells from aged mice mounted a significantly reduced proliferative response to their cognate antigen compared to identically treated young cells when unstimulated BMDC were used as APC (Figure 3A). When the BMDC were pre-activated overnight with CpG the proliferative response of the co-cultured aged naïve CD4 T cells was significantly enhanced. To better analyze the effects of CpG preactivation of BMDC on the kinetics and magnitude of the aged naïve CD4 T cell response, the fold expansion over the input number of young and aged HNT CD4 T cells on each day of a five-day culture was determined. Presenting T cell numbers as “fold expansion” allows us to better analyze combined results of multiple experiments and compare effects of different experimental treatments. Expansion is a reflection of not only the division of the activated cells, but also their survival and is physiologically relevant. We noted that aged cells activated by antigen plus untreated BMDC demonstrated a marked defect in expansion on days 4–6 compared to identically treated young cells (Figure 3B), and that activated aged cells produced only about one-half of the amount of IL-2 produced by their young counterparts (Figure 3C). In contrast, when antigen was presented by CpG-stimulated BMDC there was a significant increase in the expansion of both young and aged naïve CD4 T cells on days 4–6 that correlated with a nearly 2-fold increase in the amount of IL-2 found in 48-hour culture supernatants (Figures 3B, C). Importantly, CpG-activated BMDC derived from the bone marrow of aged BALB/c mice were equally capable of inducing the enhanced antigen-driven expansion of young and aged naïve HNT CD4 T cells (Figure 3D). Given that both young and aged BMDC were functionally activated to the same extent by TLR stimulation and the relative greater availability of young mice, we used young BMDC for the remainder of the studies.

Figure 3.

Antigen presentation by CpG activated BMDC enhances the antigen driven response of aged naïve HNT Tg CD4 T cells. Sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells were cultured with HA_126–138 plus either non-stimulated or CpG stimulated BMDC. A, [³H] TdR incorporation assay 3 days after the initiation of culture. Data are representative of two separate experiments. B, Kinetics of fold expansion over input number of T cells during 5-day culture. Combined data from 3 separate experiments. C, Supernatant levels of IL-2 measured 48 hours after initiation of culture. Combined data from 5 separate experiments. D, Fold expansion by day 5 of young and aged T cells cultured with non-stimulated versus CpG-stimulated young and aged BMDC. Combined data from two separate experiments. B, * indicates p<0.05 for aged no stim. versus young no stim., and aged no stim. versus aged + CpG for days 4–6. All other panels, * indicates p<0.05 between indicated groups.

We screened a number of other TLR agonists for their abilities to activate BMDC to enhance the expansion of aged naïve CD4 T cells. We observed that, similar to CpG, activation of the immature BMDC with either PolyI:C, Pam₃CSK₄, LPS, or loxoribine, induced significantly greater antigen-driven expansion of aged naïve CD4 T cells (Figure 4A). At this time point, only BMDC activated by PolyI:C and CpG induced a significantly greater level of expansion than the already strong proliferative response of young CD4 T cells activated by non-stimulated BMDC (Figure 4A). The differential effects of antigen presentation by TLR stimulated APC on young and aged responses may provide future clues as to what is defective in the aged naïve T cell.

Figure 4.

Antigen presentation by BMDC activated by a panel of TLR agonists boosts the aged naïve CD4 T cell response. Sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells were cultured with HA_126–138 plus either non-stimulated BMDC or BMDC stimulated with the indicated TLR agonist. A, Fold expansion of young and aged CD4 T cells over input number of T cells by day 5 of culture. Data is combined from 3 separate experiments. *indicates p<0.05 for TLR treated group compared to appropriate young or aged non-stimulated control. B, CFSE-monitored division of aged T cells on day 2 of culture and C, expression of CD25, CD62L, and CD162 by aged T cells on day 4 of culture with HA_126–138 plus non-stimulated (shaded) versus either CpG, Poly I:C, or Pam₃CSK₄-stimulated BMDC (heavy line). Data are representative of 2 similar experiments.

In further studies we use CpG, which was often most effective, and PolyI:C and Pam₃CSK₄ because of the different T cell cytokine responses they have been shown to induce (30, 55) and because they work through distinct TLR pathways, giving us a greater opportunity to identify a pathway that may be shared with humans. We observed that BMDC activated by any of the three TLR ligands induced more pronounced antigen-stimulated division of aged CD4 T cells as monitored by the loss of the intracellular dye CFSE after 48 hours of culture (Figure 4B). Similar to aged cells, a slightly greater number of young naïve CD4 T cells activated by TLR-activated BMDC had undergone additional rounds of division compared to cells activated by non-stimulated BMDC (Supplemental Data Figure 3).

Phenotypic analysis of aged CD4 T cells cultured for four days with TLR-activated BMDC demonstrated increased expression of the IL-2 receptor α chain (CD25, Figure 4C), an activation marker otherwise down-regulated on effectors from aged naïve CD4 T cells (11, 14, 16). This result was specific for responding aged CD4 T cells, as no increase in the expression of CD25 was noted on young CD4 T cells activated by TLR activated BMDC (Supplemental Data Figure 3B). We observed no difference in the expression of CD62L and CD162 between by aged effectors cultured with TLR stimulated versus non-stimulated BMDC (Figure 4C). In comparison, young cells activated with TLR stimulated BMDC had slightly lower levels of CD62L compared to those activated with non-stimulated BMDC. Taken together, these data demonstrate that DC directly stimulated by the TLR ligands CpG, polyI:C, and Pam₃CSK₄ act as superior APC to boost the cognate antigen-driven expansion of aged naïve CD4 T cells.

Aged CD4 T cells activated by TLR-stimulated BMDC exhibit increased survival and Bcl-2 expression

The level of expansion of responding antigen-specific CD4 T cells is determined both by the extent of division of responding cells and by the survival of the divided progeny. Thus, we asked whether or not aged versus young naïve HNT CD4 T cells showed equivalent survival during the antigen-driven response and how this survival was influenced when TLR-stimulated BMDC were used as APC. We harvested the responding cells on days four through six of culture and examined both the number of recovered live cells and the fraction of dead cells by staining with 7-AAD, a dye that intercalates into double-stranded nucleic acids and is normally excluded by viable cells. We found that compared to young cells, the aged naïve CD4 T cells expanded less in response to antigen presented by non-stimulated BMDC, and that there was also a significant increase in the percentage of dead cells in the aged CD4 T cell effector populations (Figure 5B). Importantly, by day 5 the 7-AAD⁺ cells from all groups were between 91%–94% CFSE^int/lo (Supplemental Data Figure 4A), indicating that the greater proportion of dead cells in the aged culture was not due to a larger fraction of cells that failed to respond to antigen and thus might be dying of neglect, but instead was a result of a greater number of cells that divided and then became apoptotic. When antigen was presented by TLR-activated BMDC, the enhanced expansion of aged CD4 T cells (Figure 5A) was accompanied by a significant decrease in the percentage of dead cells in the aged CD4 T cell cultures (Figure 5B). Similar overall trends were noted with young CD4 T cells as well, in that antigen presentation by TLR-activated BMDC enhanced the expansion of young cells and decreased the percentage of dead cells found in the culture (Supplemental Data Figures 4B, C).

Figure 5.

Enhanced survival of aged CD4 T cells activated by TLR-stimulated DC correlates with increased Bcl-2 expression. Sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells were cultured for with HA_126–138 plus either non-stimulated or BMDC stimulated with the indicated TLR. Fold expansion over input number of T cells (A), and percentage of dead cells in culture as determined by 7 AAD staining (B) on days 4–6 of culture. C, MFI of intracellular Bcl-2 staining on indicated CD4 T cells on day 5 of culture. Data are representative of 2 experiments. A, B, # indicates p<0.05 for aged-no stimulation versus young-no stimulation. A, B and C, * indicates p<0.05 for aged TLR treated groups versus aged no stimulation control.

We also examined expression of Bcl-2, an anti-apoptotic molecule that can promote the viability of T cells. The level of Bcl-2 in aged cells was higher when TLR-activated versus non-treated BMDC had been used as APC, suggesting that the enhanced survival of the aged CD4 T cell effectors might be the result of factors that induce anti-apoptotic proteins that actively protect the responding cells (Figure 5C). Bcl-2 staining of young cells activated by TLR-stimulated versus non-stimulated BMDC revealed an overall increase in the TLR-treated groups, however, this increase was not statistically significant (Supplemental Data Figure 4D).

IL-6 production by TLR-activated BMDC is specifically increased upon cognate CD4 T cell interaction

TLR stimulation is known to induce DC to produce higher levels of multiple cytokines that directly impact T cell function. We hypothesized that pre-activated DC might retain their ability to produce greater levels of cytokines and that they might be reactivated to do so during cognate interaction with the CD4 T cells. We measured the accumulation of cytokine in the BMDC:aged CD4 T cell cultures when the BMDC had been pre-activated or not with TLR agonists. The results from an initial screen of day two supernatant for a wide range of cytokines revealed that the levels of IL-12p70, MIP1β, TNFα, and RANTES were only slightly or not significantly altered by CpG pre-activation of the BMDC (Figure 6A). In contrast, the low levels of IL-10 and IL-17 were increased several fold, and levels of KC and IL-6 were dramatically higher in co-cultures with CpG-activated BMDC. Interestingly, the amount of IFNγ in the aged CD4 T cell cultures was significantly less when antigen was presented by TLR-activated BMDC, a finding that may reflect the increase in IL-6, which actively reduces IFNγ production by CD4 T cells via the up-regulation the suppressor of cytokine signaling-1 (SOCS-1) protein (56). This finding is in contrast to the in vivo data presented in Figure 2C, which illustrates elevated serum levels of IFNγ four hours after TLR agonist injection. This difference is likely due to the fact that the main source of IFNγ in Figure 6A is antigen activated CD4 T cells, while in vivo IFNγ secreted in response to TLR stimulation is likely to be derived from multiple cells including activated NK or other innate cells. Overall, similar observations in cytokine levels were found in the young T cell cultures with CpG-activated BMDC at this time point (Supplemental Data Figure 5A).

Figure 6.

Enhanced IL-6 production by TLR activated BMDC during coculture with CD4 T cells. Sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells were cultured with HA_126–138 plus either non-stimulated or CpG-stimulated BMDC. Supernatant levels of the indicated cytokines on day 2 of culture (A), and of IL-6 on days 2–5 of culture (B) were determined. C, Amount of IL-6 and TNFα on day 2 of culture of aged naïve CD4⁺ HNT Tg T cells cultured with HA_126–138 plus either non-stimulated BMDC, or BMDC stimulated with the indicated TLR ligands. D–G, Naïve HNT Tg CD4 T cells were cultured with HA_126–138 plus either non-stimulated or CpG and PolyI:C stimulated BMDC labeled with CellTrace^TM Violet for 24 hours with brefeldin added during the last 5 hours. D, E Representative histograms of IL-6 intracellular staining of gated (D) CellTrace^TM Violet labeled BMDC or (E) young CD4 T cells. F, G Percent of indicated cells positive for intracellular IL-6 in cultures containing either non-stimulated or CpG and PolyI:C stimulated BMDC and either young (F) or aged (G) T cells. All culture supernatant cytokine analyses performed by cytokine bead array. The cytokine screen in A was performed on supernatant from a single experiment, and the cytokines IL-1, IL-4, IL-5, IL-13, and MCP-1 were not detected above background at this time point (data not shown). Data in B and C are representative of 2–3 similar experiments. * indicates p<0.0001 for TLR treated group compared to non-stimulated control.

The increase in IL-6 was of particular interest because it is known to increase the survival of primed CD4 T cells (57), and it is also one of a trio of cytokines (including IL-1 and TNFα) previously shown to enhance the aged CD4 T cell response in vivo (12). Figure 6B shows that supernatant levels of IL-6 remained at least 5–20 fold higher from days two through five in those cultures containing aged naïve CD4 T cells and CpG-activated BMDC versus BMDC with no pre-activation. In addition, greater than 10-fold increases in levels of IL-6 were also found in day two (Figure 6C) and day five (data not shown) supernatants from aged CD4 T cells cultured with either PAM₃CSK₄- and PolyI:C-activated BMDC. Supernatant from 2-day cultures that contained the equivalent number of pre-activated BMDC without naïve CD4 T cells did not contain significant IL-6 (Figure 6C). TNFα production was also seen in the co-cultures and required CD4 T cells, but it was not enhanced by TLR agonist pre-activation of the BMDC. Levels of IL-6 were also elevated in the day two through five supernatant of young cells cultured with CpG-activated BMDC, and they were similarly elevated in the co-cultures with PolyI:C and PAM₃CSK₄-activated BMDC (Supplemental Data Figures 5B, C).

To determine whether the BMDC or the CD4 T cells were the principal producers of IL-6 in culture, naïve HNT Tg CD4 T cells were co-cultured BMDC pre-labeled with CellTrace^™ Violet-plus antigen for 24 hours, with brefeldin added during the final five hours. Intracellular cytokine staining for IL-6 was then performed and BMDC and CD4 T cells were differentiated based upon the presence of the CellTrace^™ label and expression of CD11c and CD11b. (Supplemental Data Figure 6). We found that BMDC that had been pre-activated by CpG and PolyI:C demonstrated a significant increase in intracellular IL-6 staining (Figure 6D). In comparison, there was no difference in IL-6 staining of CD4 T cells cultured with either non-stimulated versus TLR-stimulated BMDC (Figure 6E). The specific increase in the production of IL-6 by TLR-stimulated BMDC in culture was seen with either young or aged naive CD4 T cells and is quantitated in Figures 6F and G respectively. These data suggest that the TLR-activated BMDC are the principal initial source of IL-6 during CD4 T cell:BMDC cognate interaction. Moreover, the data in Figure 6C illustrating little IL-6 production in the absence of T cells suggest that IL-6 production by BMDC in the absence of further TLR agonist stimulation requires induction by the cognate CD4 T cell:BMDC interaction.

Antigen presentation by TLR-activated splenic DC enhances the aged naïve CD4 T cell response

BMDC are thought to represent immature myeloid DC, and their immaturity could potentially make them more dependent on TLR signaling than in situ DC. To evaluate whether splenic DC would also become improved APC for naïve aged CD4 T cells, we determined whether antigen presentation by TLR-activated splenic DC was similarly capable of enhancing the aged CD4 T cell response. As illustrated in Figure 7, both young and aged naïve HNT CD4 T cells underwent a significantly greater level of expansion when placed into culture with cognate antigen and splenic DC activated by CpG and PolyI:C versus non-stimulated splenic DC. In addition, similar to the experiments with BMDC, the splenic DC activated by TLR ligands produced elevated levels of IL-6 shortly after the initiation of co-culture with the young or aged naïve CD4 T cells. These data demonstrate a similar capacity of the bone derived DC and ex vivo splenic DC to enhance the aged CD4 T cell response when pre-activated by TLR ligands.

Figure 7.

Antigen presentation by splenic DC activated by TLR agonists boosts the aged naïve CD4 T cell response. Sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells were cultured with HA_126–138 plus either non-stimulated or CpG and PolyI:C stimulated splenic DC. A, Fold expansion of young and aged CD4 T cells over input number of T cells by day 5 of culture. B. Percent positive IL6 producing splenic DC after 24 hours of co-culture with aged or young CD4 T cells where splenic DC were treated with or without TLR ligands. *indicates p<0.05 for TLR treated group compared to young or aged non-stimulated control.

IL-6 blockade diminishes the adjuvant activity of TLR ligands

The high levels of IL-6 produced by the TLR-activated BMDC suggest that this cytokine might play a key role in enhancing the naïve CD4 T cell response. To test whether IL-6 was necessary for the enhanced response, aged naïve HNT CD4 T cells were cultured with either CpG-, PolyI:C- or Pam₃CSK₄-pretreated BMDC in the presence of anti-IL-6 blocking Ab, and for comparison anti-TNFα blocking Ab. We found that addition of blocking Ab to IL-6, but not to TNFα at the initiation of culture significantly diminished the enhancing effect of TLR activation of BMDC on T cell expansion and survival (Figures 7A, B). The enhanced expansion and survival of young naïve CD4 T cells activated by CpG-stimulated BMDC was also blocked by the addition of blocking Ab to IL-6 but not to TNFα (Supplemental Data Figure 7). Importantly, including both IL-6 and TNFα blocking antibodies together showed no synergistic effects compared to blocking IL-6 alone (data not shown).

Based upon the blocking experiments above, we tested whether or not the addition of IL-6 alone during the activation of naïve aged CD4 T cells was sufficient to enhance their expansion and since multiple inflammatory cytokines were made, we also tested whether or not supernatant from (day 2) aged HNT CD4 T cell:TLR-activated BMDC cultures was able to enhance aged CD4 T cell expansion. In this experiment aged naïve HNT CD4 T cells were stimulated with plate-bound anti-CD3 and anti-CD28. We had previously shown that stimulation using plate-bound anti-CD3 and anti-CD28 revealed similar defects in the aged naïve CD4 T cell response equivalent to those seen with antigen and APC (58). This mode of stimulation allowed us to assess the effects of IL-6 without the complication of additional cytokines that would be elaborated by co-culture with BMDC. As seen in figure 8C, the addition of titrated amounts of recombinant IL-6 (from 10–90 ng/ml, 24), did not enhance the expansion of aged CD4 T cells. In contrast, the addition of the day 2 supernatant significantly enhanced the expansion of the aged CD4 T cells, suggesting that IL-6 is necessary but not sufficient and that other inflammatory cytokines produced during naïve CD4 T cell:TLR activated DC co-culture might also play key roles in enhancing expansion of antigen-specific CD4 T cells.

Figure 8.

IL-6 is required for the TLR-mediated enhancement of the aged naïve CD4 T cell response. A and B, Sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells were cultured with HA_126–138 plus either non-stimulated or TLR-stimulated BMDC with or without either anti-IL-6, anti-TNFα or irrelevant isotype control. A, Fold expansion over input number of T cells, and B, percentage of dead cells on day 5 of culture as determined by 7 AAD staining. C, Fold expansion of sorted young and aged CD4⁺Vβ8.3⁺CD62L^hiCD44^lo HNT Tg T cells stimulated by plate bound anti-CD3 and anti-CD28 with or without the addition of recombinant IL-6 at the indicated concentrations or 48 hour supernatant (1:7 final dilution) from aged HNT Tg T cell + HA_126–138 + TLR-activated BMDC cultures. Data from A, B are representative of 2 similar experiments. Data in C are from a single experiment. A, B *indicates p<0.05 for blocking antibody treated samples compared to isotype antibody treated control of the same TLR treatment. C, * indicates p<0.05 for aged plus supernatant compared to aged alone.

Together, these findings indicate that pre-activation of BMDC with TLR agonists results in the production of IL-6 when the BMDC act as APC for aged naïve CD4 T cells, and that this IL-6, likely working in concert with some other soluble factor(s), is necessary for the enhanced expansion and survival of the responding cells as they become effectors.

Discussion

Here we report, that pre-activation of DC through multiple TLR endows them with an enhanced ability to promote naïve CD4 T cells responses. In particular, the otherwise poor responses of aged naïve CD4 T cells are improved, resulting in greater expansion of the aged T cells due in part to the reduction of apoptosis that otherwise occurs preferentially in the aged T cells and limits their expansion. The improved aged CD4 T cell response is dependent on IL-6, whose production in cognate cultures is much enhanced by the TLR preactivation. The DC are a major source of IL-6 in the co-culture. Addition of IL-6 alone cannot replace the TLR pre-activation, but supernatants from co-cultures have some enhancing ability, suggesting synergy among the factors produced. These results suggest that strategies to specifically target DC APC with TLR adjuvants might improve the efficacy of vaccines without the need for systemic TLR administration.

Although naïve CD4 T cells show severe age-associated defects in vitro and in vivo in response to peptide and protein antigens, the fact that addition of proinflammatory cytokines can improve the response of such T cells and their ability to provide help for B cells, suggested that TLR-agonists might act to improve aged naïve CD4 T cell responses by inducing production of such cytokines by APC. A recent paper by Maue et al (43) demonstrated that the Poly I:C was an effective adjuvant in vivo and that it increased the helper activity of aged CD4 T-cells. However, studies of the mechanism of TLR ligand adjuvant activity in vivo are limited in their ability to pinpoint cell types activated by TLR administration and the mechanisms by which they contribute to the enhanced T cell dependent B cell response. In the in vitro model presented here, DC are first pre-activated with the TLR ligand before being extensively washed so that TLR activation is restricted to the APC and we can avoid the influence of direct engagement of TLR expressed by T cells themselves. The pre-activated DC are co-cultured with antigen plus young versus aged sorted naïve HNT transgenic T cells. In this model, the untreated BMDC represent those functionally immature DC found underneath the epithelial surfaces of the body. In vivo, upon stimulation by cytokines or conserved pathogen motifs these cells migrate to the draining lymph nodes where they present antigen to naïve CD4 T cells. This transformation includes the down-regulation of pathogen receptors, and the up-regulation of molecules involved in T cell activation (MHC class II, CD80, CD86, IL-12). Our results suggest that immature DC activated this way, via either TLR 2, 3, or 9, are highly potent APC with the capacity to provide strong enough activation and costimulation signals such that the defects in expansion normally seen in aged naïve CD4 T cells are largely overcome. We note this does not imply that the specific defective pathways are corrected, merely that enhancement is sufficient that function is improved.

Because the addition of supernatant from CD4 T cell/TLR-stimulated BMDC co-cultures was sufficient to significantly enhance the expansion of aged CD4 T cells, we suggest that many of the effects of TLR preactivation are likely to be due to elevated cytokines. IL-6 was a likely candidate because it was one of a small number of cytokines that was dramatically up-regulated in culture and it is known to impact naïve CD4 T cell responses. This hypothesis was strengthened by the fact that the addition of anti-IL-6 blocking antibodies (but not anti-TNFα) prevented the TLR-activated BMDC from boosting the aged HNT CD4 T cell response.

Comparison of the responses of young versus aged naïve cells stimulated with untreated DC suggests that increased apoptosis of dividing aged T cells is at least partly responsible for their poor expansion. In addition to enhancing the proliferation of aged effectors, the TLR ligand treatment of APC promoted activated T cell survival and slightly increased expression of Bcl-2, as in previous studies of young CD4 T cells (59). We found that the enhanced survival is also dependent on IL-6 and is thus likely to be due to the increased IL-6 production that results from the TLR ligand stimulation of the DC, consistent with previous studies demonstrating that the addition of IL-6 increases the survival of primed CD4 T cells (57). The results from the current study suggest a novel additional role for IL-6, as a key factor produced during the cognate interaction between a CD4 T cell and a TLR-activated DC that is capable of enhancing the response of aged naïve CD4 T cells. The intracellular staining presented in Figure 7 suggests that the TLR activated BMDC were the principal initial producers of IL-6 in the CD4 T cell-DC co-cultures. In this regard, it is significant to note that supernatant from two-day cultures that contained TLR-activated BMDC alone in numbers equivalent to those in co-cultures, did not contain significant IL-6 (Figure 6). This suggests that IL-6 production by BMDC in the absence of further TLR agonist stimulation requires the CD4 T cell:BMDC interaction. Such an induction of APC function could potentially be mediated through CD40:CD40L interactions (60).

The study of TLR and their natural agonists has revealed that ligation of different TLR may trigger differential cytokine production in the same cells, or result in different cytokines produced by different types of APC (61). In addition, several reports suggest that response to TLR stimulation and the patterns of TLR expression are different on human versus mouse APC (62, 63). We were impressed by the consistency of response we saw to the different TLR agonists used in these studies. Thus, despite the fact that CpG, PolyI:C and PAM₃CSK₄ mimic both viral and bacterial components and interact with different TLR, including those expressed on the cell surface and within endosomes, we found that all three agonists induced comparable improvements in aged naïve CD4 T cell expansion. In each case, this was due in part to less apoptosis and was dependent on IL-6. NF-κB, a transcription factor responsible for IL-6 production, is activated by most TLR downstream of the MyD88 adaptor protein. Activation via TLR3, which does not recruit MyD88, can still result in the activation of NF-κB via the recruitment of TNF receptor-associated factor 6 (TRAF-6). Therefore, it is likely that the activation of BMDC to enhance aged naïve responses is part of a shared pathway induced by many TLR agonists, thus, making it more likely that a similar pathway will occur in the human. It does not appear that the results from the current study were influenced by contaminating endotoxin, since a number of steps were taken to rule out contamination of the different TLR agonist preparations, including use of control non-CpG ODNs, LAL testing of endotoxin levels and stimulation of TLR4 KO BMDC with the panel of TLR ligands (see Materials and Methods).

Future experiments are planned to further dissect the mechanism by which IL-6 may help to rescue the aged response. Like IL-12, IL-6 has been shown to be important for the clonal expansion and differentiation of primed cells (57, 64). However, whereas IL-12 promotes Th1 responses, particularly through the induction of IFNγ (64), IL-6 promotes Th2 responses via the induction of IL-4, and the active suppression of IFNγ via the upregulation of SOCS-1 (56). Furthermore, IL-6 is necessary for polarization of naïve CD4 T cells in mice and humans to become Th17 polarized cells. IL-6 has also recently been shown to induce the production of IL-21 by activated CD4 T cells (65). This finding is especially important since the production of IL-21 by CD4 T cells is now understood to be essential for B cell activation, expansion and PC differentiation (66), as well as playing a role in polarization of T cells. Thus, we are especially interested in whether augmentation of IL-21 production by IL-6 produced by TLR activated BMDC, will help to overcome the observed defects in aged CD4 T cell cognate B cell help.

It will be interesting to directly compare the efficacy, and to determine if shared mechanisms are responsible, for alternate protocols that enhance aged naïve CD4 responses including the addition of either IL-2 (11), or a combination of inflammatory cytokines (IL-6, TNFα and IL-1), which have also been shown to enhance the expansion of aged naïve CD4 T cells in vitro and in vivo (12). The effect of these treatments appeared to be primarily due to the increased proliferation of antigen activated T cells. Systemic cytokine treatments are probably not appropriate for elderly patients, since they may cause unwanted nonspecific, systemic effects. Localized administration of TLR ligands, potentially in conjugation with antigen, has the potential to limit such nonspecific effects. Ex vivo treatment of autologous APC would further limit bystander exposure and the requirement for cognate CD4 T cell:APC interaction for the production of IL-6 by the pre-activated BMDC, as shown in this study, may help to direct cytokines primarily to antigen specific T cells reducing unwanted systemic effects.

In conclusion, the wide-range of functional defects that accumulates with age in the naïve CD4 T cell compartment result in the reduced immune response to vaccines in the aged, thus limiting vaccine efficacy in an already highly susceptible population. The data presented here suggests that the use of TLR ligands as vaccine adjuvants acting directly on aged APC is effective in vitro and probably in vivo where it is seen in intact aged mice. Through definitive in vitro models, we have identified a likely mechanism by which TLR stimulation acts to enhance aged CD4 T cell proliferation and survival. Central to this mechanism is the increased production of the inflammatory cytokine IL-6 by TLR-activated APC during their cognate interaction with antigen-specific naïve CD4 T cells, which, perhaps along with additional factors, enhances the expansion and survival of responding aged T cells. This effect is remarkable in that it restores the magnitude of the response of aged naïve CD4 T cells to levels comparable to those seen with young cells.