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. Author manuscript; available in PMC: 2015 May 15.
Published in final edited form as: J Immunol. 2014 Oct 10;193(10):5056–5064. doi: 10.4049/jimmunol.1301723

4-1BB ligand activates bystander dendritic cells to enhance immunisation in trans1

Douglas C Macdonald *, Alastair Hotblack *, Saniath Akbar, Gary Britton, Mary K Collins @,+, William C Rosenberg #,+
PMCID: PMC4255227  EMSID: EMS60379  PMID: 25305314

Abstract

Expression of the co-stimulatory receptor 4-1BB is induced by T cell receptor recognition of antigen, while 4-1BB ligand is highly expressed on activated antigen presenting cells. 4-1BB signalling is particularly important for survival of activated and memory CD8+ T cells. We wished to test whether co-expression of antigen and 4-1BBL by dendritic cells (DC) would be an effective vaccine strategy. We therefore constructed lentiviral vectors (LV) co-expressing 4-1BBL and influenza nucleoprotein (NP). Following subcutaneous immunisation of mice, which targets DC, we found superior CD8+ T cell responses against NP and protection from influenza when 4-1BBL was expressed. However, functionally superior CD8+ T cell responses were obtained when two LV were co-injected, one expressing 4-1BBL, the other expressing NP. This surprising result suggested that 4-1BBL is more effective when expressed in trans, acting on adjacent DC. We therefore investigated the effect of LV expression of 4-1BBL in mouse DC cultures and observed induced maturation of bystander, untransduced cells. Maturation was blocked by anti-4-1BBL antibody, required cell-cell contact and did not require the cytoplasmic signalling domain of 4-1BBL. Greater maturation of untransduced cells could be explained by LV expression of 4-1BBL causing down-regulation of 4-1BB. These data suggest that co-expression of 4-1BBL and antigen by vaccine vectors that target DC may not be an optimal strategy. However, 4-1BBL LV immunisation activates significant numbers of bystander DC in the draining lymph nodes. Transactivation by 4-1BBL/4-1BB interaction following DC/DC contact may therefore play a role in the immune response to infection or vaccination.

Keywords: co-stimulation, vaccine, dendritic cell

Introduction

When naïve T cells are stimulated by antigen, engagement of CD28 by its ligands CD80 or CD86 (termed co-stimulation) lowers the threshold for activation (reviewed in[1]). T cell activation then induces expression of members of the tumour necrosis factor receptor (TNFR) family including 4-1BB, which transmits activation and survival signals during subsequent effector and memory T cell stages (reviewed in[2]).

The effects of 4-1BB are most pronounced in CD8+ T cells, for example systemic administration of agonistic anti-4-1BB antibody to mice expands CD8+ T cells more than CD4+ T cells[3]. Natural T-regulatory CD4+ T cells are an important exception to this, since they constitutively express 4-1BB[4] as are a new subset of cytotoxic CD4+ T cells[5]. This selectivity for CD8+ T cell stimulation may be due to greater expression of 4-1BB on activated CD8+ compared with CD4+ T cells[6]. During infection of 4-1BBL deficient mice, with influenza or LCMV, the CD8+ T cell response is impaired[7]. Also when human T cells are expanded in vitro with HIV[8] or influenza [9] antigens, 4-1BB triggering increases expansion of CD8+ T cells with polyfunctional cytokine secretion and higher perforin and granzyme A expression.

Duration and severity of infection influence expression of both 41BB on T cells and 4-1BBL on antigen presenting cells. For example, 4-1BBL expression is only significantly up-regulated in lung monocytes when mice are infected with the lethal influenza A/PR8 infection and not the milder A/X31[10]. In such A/PR8 infection, 41BBL is redundant for the primary response but remains essential for secondary T cell expansion upon antigen re-encounter[10]. 4-1BBL on antigen presenting cells is capable of “reverse signalling”. Thus 4-1BBFc or anti-4-1BBL mAb induces proliferation of monocytes, and stimulates cytokine secretion[11]. DC maturation can also be stimulated through 4-1BBL[12].

Activated DC can also express 4-1BB[13] bringing an extra level of complexity. Stimulation of 4-1BB on DC results in secretion of cytokines [13, 14]. Administration of the agonistic anti-4-1BB antibody to RAG-1 knockout mice improves the ability of splenic DC from these mice to stimulate proliferation of T cells[13]. This complicates studies examining T cell responses to 4-1BB, for example after stimulation by agonistic antibody, since this may occur both directly through T cell 4-1BB and indirectly through DC activation.

The important role of 4-1BB in the CD8+ T cell effector and memory response has led to a number of attempts to harness 4-1BB for immunotherapy or vaccination. Agonistic 4-1BB antibody promotes effective anti-tumour CD8+ T cell responses in mice[15, 16] and clinical trials of this approach have been initiated (reviewed in[17]). Engineering tumour cells to express 4-1BBL also inhibits tumour growth and induces tumour-specific T cells[18]. This approach of localised 4-1BBL expression, rather than systemic agonistic antibody, has the potential to focus the immune stimulation on T cells that recognise the tumour antigens. Similarly, 4-1BBL has also been expressed, along with antigen, for immunisation against infectious disease. For example, superior CD8+ T cell responses against HIV Gag and Pol epitopes are induced by 4-1BBL expression in poxvirus or DNA vaccines in mice[19-21]. An intranasal adenoviral vector (AV) co-expressing 4-1BBL and influenza NP generates superior lung CD8+ T cell responses and protection against PR8 challenge than one expressing NP alone[22]. This 4-1BBL AV also enhances human CD8+ T cell recall responses to NP, giving greater expansion of NP peptide-specific T cells with up-regulated granzyme-A, perforin and cytolytic activity[22].

Haematopoeitic cells have been stably transduced with a lentiviral vector (LV) expressing 4-1BBL to generate a stimulatory antigen-presenting cell line capable of long-term T cell expansion in vitro for immunotherapy[23]. However, the potential of LV expressing TNFR family ligands as vaccines has not previously been explored. LV are currently being tested as vaccine vectors in an initial clinical trial in HIV infected vaccine recipients (reviewed in [24]). The ability of LV to transduce non-dividing antigen presenting cells in situ[25] and their relatively low intrinsic immunogenicity[26] make them ideal vectors to explore the potency of adjuvants[27, 28]. In addition, the long-term expression persistence of antigen presentation achieved by LV[29, 30] might benefit T cell memory, since tonic 4-1BB stimulation may sustain memory populations independently of antigen. We therefore constructed a LV expressing 4-1BBL and NP and found that this gave superior CD8+ T cell responses against NP and greater protection against lethal influenza challenge than LV expressing NP alone. Our initial hypothesis was that 4-1BBL and NP expressed by the same antigen-presenting cell would be most effective. However, when we examined the mechanism of action of 4-1BBL expressed by LV we found that at least part of its effect is to activate adjacent, bystander DC by triggering 4-1BB.

Materials and Methods

Lentiviral production

Lentiviral vectors were derived from pHRSIN-CSGW [31] by insertion of a second promoter and transgene downstream of the WPRE element. Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped lentivectors were produced by co-transfection of the vector with pCMVR8.91 (Gag/pol) and pMDG (VSV-G envelope) as previously described [32]. A lentivector co-expressing a shRNA targeting 4-1BB together with NP was constructed as described previously [41]. Supernatants were concentrated by ultracentrifugation at 24,000 rpm through a 20% sucrose cushion. Titres were measured by reverse transcriptase assay (Roche Diagnostics, UK) and quantitative TaqMan PCR (Applied Biosystems, UK).

Preparation and transduction of DC

Murine bone-marrow derived DCs were prepared as described previously[33] and transduced on day 4 of culture in RPMI 1640 5% FCS and 50 ng/ml GM-CSF at MOIs ensuring >80% transduction. Control DC were matured with 100 ng/ml LPS. 3 days after transduction cells were harvested for staining and analysis by FACS. After Fc-receptor blockade, cells were stained with anti-CD11c-APC (eBiosciences) and either anti-mouse PE-conjugated ICAM-1, CD40, (eBioscience), CD80, CD86 or MHC II (BD Biosciences). Antibodies against 4-1BBL, 4-1BB and blocking antibody TKS-1 were from eBiosciences. Cells were gated for analysis on GFP+CD11c+ for transduced groups and CD11c+ for control groups.

Immunisation and influenza challenge

Female BALB/c mice, 6 – 8 weeks old, were purchased from Charles River Laboratories and kept in pathogen-free conditions. A dose of 50 ng RT was given for subcutaneous immunisation. Intranasal challenges with influenza virus was performed under ketamine and xylazine anesthesia by inoculation of 20 μl of viral suspension into each nostril. Following challenge, mice were weighed daily from Day 3 onwards and clinical signs of disease were scored. Mice were sacrificed if weight loss exceeded 25%.

The influenza strain used was mouse-adapted A/PR/8/34 (a kind gift from Dr Mike Whelan, UCL). Virus was titrated by plaque assay in MDCK cell layers inoculated with 100 μl serially diluted samples and incubated for 3 days after overlay with 0.6% agarose (oxoid) in DMEM with 2 μg/ml trypsin (Worthington). A dose of 2×LD50 corresponded to 2500 PFU per mouse by plaque assay. This gave 100% lethality in control mice.

Immunoanalysis

Cells isolated from spleen or lymph node were either stained immediately to quantify and phenotype NP-specific T cells (NP147-155 pentamer-PE (ProImmune, UK), anti-CD8-APC, (eBioscience) anti-GzmB-APC, alternatively stimulated overnight with relevant peptide and brefeldin A (1 μg/ml) in the last 5 hours before staining followed by permeabilisation for intracellular cytokine staining (anti-GzmB-APC, anti-IFNγ-FITC). For re-stimulation of CD8+ T cell responses in culture, the NP147-155 TYQRTRALV peptide, a H2Kd restricted CD8 epitope was used. For re-stimulation of CD4 T cell responses, the peptide NP55-78 RLIQNSLTIERMVLSAFDERRNKY was used. In addition to intracellular cytokine staining analysis of lung CD4 T cells, splenocytes were re-stimulated for 4 days in the presence or absence of peptide NP55-78 in RPMI 5% FCS. Supernatants were then harvested and frozen for cytokine analysis by cytometric bead array (TH-1/TH-2 FlowCytomix kit, eBiosciences).

Statistical analyses

All data were analysed using the GraphPad Prism v5.0 statistical software package. Statistical tests applied to each data set are indicated in the relevant figure legend.

Results

Engineering dendritic cells to express 4-1BBL

In order to constitutively express 4-1BBL on antigen presenting cells, lentiviral vectors (LV) were constructed to express 4-1BBL together with either green fluorescent protein (GFP) (Figure 1A). Expression of mouse 41BBL was detected by surface staining of transduced 293T cells or mouse bone marrow-derived mouse dendritic cells (DC) on day 3 after transduction (Figure 1B). Endogenous 4-1BBL was not detected on mouse DC after 4 days in culture, or on mouse DC transduced with a control LV expressing GFP alone (Figure 1C). Dose-dependent increasing expression of 41BBL was detected after transduction of mouse DC with increasing amounts of 4-1BBL-GFP LV (Figure 1C).

Figure 1. Engineering mouse dendritic cells to express 4-1BBL.

Figure 1

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(A) A lentiviral vector designed to express mouse 4-1BBL together with GFP or NP. (B) Expression of 4-1BBL in 293T cells and mouse bone marrow-derived DC (MuDC) after transduction with 4-1BBL-GFP. (C) Dose-dependent expression of 4-1BBL (black) and GFP (grey) in mouse DC after transduction with 4-1BBL-GFP

4-1BBL enhances NP-specific T cell responses and influenza protection

When LV encoding antigens are injected subcutaneously they predominantly transduce DC, which are necessary and sufficient for initiating a T cell response to LV encoded antigen[34-36]. In order to determine whether 4-1BBL enhanced T cell responses against co-encoded antigen, Balb/c mice were vaccinated subcutaneously with 4-1BBL-NP or Null-NP or saline and sacrificed 14 days later for splenocyte analysis (Figure 2A). CD4+ and CD8+ T cell responses were assessed by IFN-gamma ELIspot after overnight re-stimulation with Class II or Class I restricted peptides respectively. This revealed significantly greater CD4+ and CD8+ T cell responses to vaccination with 4-1BBL-NP than Null-NP (Figure 2B). Splenocytes were also re-stimulated in vitro for 4 days with Class II restricted peptide and supernatants analysed by cytometric bead array for a panel of 10 cytokines including IFNγ, TNFα, IL-4, IL-10, IL-17, GMCSF, IL-1α, IL-5 and IL-6. Significantly greater concentrations of TNFα, IFNγ and GMCSF were found in supernatants in responses to class II restricted peptide re-stimulation in splenocytes cultures from 4-1BBL NP vaccinated mice compared with mice vaccinated with Null-NP (Figure 2C). This resembles the findings of Li et al who demonstrated increased IFNγ and GMCSF secretion by T cells (and reduced Th-2 cytokine secretion) in tumour-draining lymph nodes following stimulation with anti-41BB antibody in vivo[37].

Figure 2. 4-1BBL enhances mouse T-cell responses against co-encoded NP.

Figure 2

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Mice were sacrificed 14 days after subcutaneous vaccination with a lentiviral vector encoding 4-1BBL and influenza NP in place of GFP, according to the schedule shown in (A). (B) CD8+ T-cell and CD4+ T-cell IFNγ ELIspot after overnight re-stimulation of splenocytes with Class I or Class II restricted NP peptide as shown. (C) Cytokine levels in the supernatants of splenocytes re-stimulated in vitro with Class II restricted peptide. Paired T test, *p=0.01-0.04; ***p<0.0009.

14 days after subcutaneous lentiviral vector immunisation, mice were challenged with a 2×LD50 dose of A/PR/8/34 and monitored for weight loss. All mice developed the severe clinical syndrome of weight loss, tachypnoea and hunched posture. However, at day 6 6/10 mice vaccinated with 4-1BBL-NP began to recover weight, which returned to normal at day 12, while 4 died. Only 2/18 mice survived in the Null-NP group although these 2 mice regained their normal weight (Figure 3). Thus 4-1BBL-NP vaccination confers a significant survival benefit upon lethal influenza challenge compared with lentiviral vector expressing NP alone.

Figure 3. 4-1BBL enhances protection of mice against lethal influenza challenge.

Figure 3

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Survival (A) and weight loss (B) of balb/c mice after subcutaneous vaccination with lentiviral vectors encoding 4-1BBL and influenza NP or influenza NP alone followed after 14 days by lethal PR8 influenza challenge. **Long-rank Test: p=0.0008.

4-1BBL enhances antigen-specific CD8 T cell function when expressed in trans

Our working hypothesis was that 4-1BBL expression on the same dendritic cells that expressed and presented NP peptides was leading to enhanced T cell responses. To test this we compared immunisation with a LV co-expressing 4-1BBL and NP with immunisation with two separate LV, one encoding 4-1BBL and the other NP. Groups of 4 mice were vaccinated subcutaneously on one side in two separate sites which drain to the inguinal lymph node, as shown in Figure 4A. In previous studies we have tracked transduced DC in the lymph node following subcutaneous injection [26, 36]. Very few DC (<1% of CD11c+ cells in the lymph node) would have been transduced with either vector at this dose, so the likelihood of dual transduction was very low. Lymph nodes and spleens were harvested 14 days after immunisation and T cell responses were then assessed.

Figure 4. 4-1BBL enhances the quality of the CD8+ T cell response when expressed in trans.

Figure 4

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Mice were immunised at 2 separate sites in the same or opposite flanks with a lentiviral vector encoding 4-1BBL and influenza NP together or with 2 separate vectors encoding influenza NP alone and 4-1BBL alone, then analysed after as described in (A). Panel B. Mice were immunised at 2 sites in the same flank then analysed after 14 days. The percentage of NP147-155 pentamer+/CD8+ cells in spleen (B, upper panel), granzymeB+/CD8+ cells in spleen (B, middle panel) and granzyme B+/CD8+ cells in the draining lymph nodes (B, lower panel) are shown. Paired T test, *p=0.01-0.04. Panel C. Lentivectors expressing NP or NP together with a shRNA targeting 4-1BB were used to transduce mouse bone marrow-derived dendritic cells which were then activated with LPS (10^g/ml) for 6 h. 4-1BB MFI was reduced from 7642 to 1937 by the shRNA. Panel D. Mice were immunised at 2 sites in the same or opposite flanks with the lentiviral vectors shown then analysed after 5 days. The percentage of granzyme B+/CD8+ cells in the draining lymph nodes from both flanks (Left or Right as indicated) is shown as the mean plus SEM. Paired T test, *p=0.01-0.04; ***p<0.0009.

CD4+ ex vivo responses were not significantly different between the 4 groups of vaccinated mice and NP147-155 pentamer responses in the spleen were equivalent between all 4 groups (Figure 4B upper). However, mice receiving 4-1BBL-GFP in trans with Null-NP demonstrated higher GzmB expression in CD8+ T cells after overnight stimulation with the CD8-restriced peptide than was observed in mice receiving 4-1BBL-NP and Null-GFP in cis (Figure 4B middle). In the lymph node, the mice receiving 4-1BBL-GFP in trans with Null-NP were the only group to demonstrate significantly greater GzmB expression upon re-stimulation (Figure 4B lower). These striking data suggested that 4-1BBL expressed on one population of DC was enhancing NP antigen stimulation of T cells by adjacent DC. To test this hypothesis we injected 4-1BBL-GFP and Null-NP on the same or opposite flanks and examined the NP response after 5 days in the draining lymph node. Figure 4D shows that injection on opposite flanks did not lead to stimulation in trans supporting the idea that direct DC contact was necessary. We then produced a lentiviral vector expressing a shRNA direct against 4-1BB together with NP, which down-regulated 4-1BB by approximately 4-fold when tested in DC cultures (Figure 4C). This vector did not respond to 4-1BBL stimulation in trans when co-injected on the same flank (Figure 4D), again supporting a mechanism of direct DC interaction.

4-1BBL activates bystander DC in vitro

DC activation by 4-1BBL has been shown to occur both by 4-1BBL reverse signalling, and by stimulation of 4-1BB. We therefore examined the expression of 4 activation markers (CD40, CD80, CD86 and ICAM-1) on mouse bone marrow-derived DC following in vitro transduction with 4-1BBL-GFP on day 3 of culture, followed by a further 4 days of culture. Figure 5 shows that transduction of these DC cultures with a control LV Null-GFP caused a modest level of activation of the GFP positive cells; we have previously shown that this was due to TLR3 and TLR7 triggering on DC by the LV leading to some activation by the LV particle alone [26]. Inclusion of the potent NFkappaB activator vFLIP caused a more marked activation as we previously described [28], in this case in the GFP positive transduced cells. Strikingly, 4-1BBL-GFP caused a marked and more pronounced DC activation, predominantly in the GFP negative, untransduced cells.

Figure 5. 4-1BBL activates bystander, untransduced dendritic cells.

Figure 5

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Analysis of four markers of DC activation at Day 4 after transduction of mouse bone marrow-derived dendritic cells with 4-1BBL-GFP, Null-GFP or vFLIP lentiviral vectors. Expression of each marker is expressed as a factor of increase relative to untransduced DC (with the dotted line at 1 indicating no increase). Analyses of paired transduced (GFP+ve) cells and untransduced (GFP-ve) cells from the same well are joined by a connecting line. T-tests comparing log(fold increase) were used to determine the p-value of observed differences between paired groups, *p=0.01-0.04, **p=0.001-0.009, ***p<0.0009, NS= not significant.

4-1BBL induced bystander DC activation is independent of reverse signalling, requires cell-cell contact and is abrogated by blocking anti-4-1BBL antibody

To investigate the role of potential reverse signalling in DC maturation, we created truncated mutant lacking the cytoplasmic N-terminal domain which includes 2 putative casein kinase II signalling regions [38]. This mutant was expressed on the cell surface to an equivalent degree as wild-type (Figure 6A). The DC activation assay revealed stronger up-regulation of activation markers in the untransduced population with the truncated 4-1BBL (Figure 6A) to the same degree as observed for the full length 4-1BBL (Figure 5).

Figure 6. 4-1BBL activates bystander dendritic cells via 4-1BB.

Figure 6

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Truncation of 4-1BBL to remove the cytoplasmic domain (4-1BBLTc) has no effect on expression level in dendritic cells transduced with 4-1BBL-GFP or 4-1BBLTc-GFP (A, upper) or transactivation of untransduced DC in the target population (A, lower). Addition of 4-1BBL transduced DC to an upper well separated from a lower well containing untransduced DC (by a 0.4um pore membrane) failed to induce activation suggesting cell-cell contact is required for transactivation(B). (C) Transactivation of untransduced DC is abrogated in the presence of anti-4-1BBL blocking antibody clone TKS-1. This occurs in both 4-1BBL WT and Tc transduced populations. Paired T test, *p=0.01-0.04, **p=0.001-0.009, ***p<0.0009.

Addition of 4-1BBL-GFP transduced DC to the upper well of transwell plates did not increase the activation of untransduced DC in the lower well, suggesting cell-cell contact is necessary for transactivation of DC by 4-1BBL, rather than a cytokine mediated mechanism (Figure 6B). Furthermore, addition of anti-41BBL blocking antibody (clone TKS-1) consistently abrogated activation of the untransduced population in these experiments, regardless of whether 4-1BBLTc-GFP or 4-1BBL-GFP was used (Figure 6C).

Taken together, these data strongly suggest that the DC activation observed in a total population of DC after transduction by 4-1BBL-GFP occurs by forward signalling to untransduced bystander DC. This presumably occurs through 4-1BB receptor expression on mouse DC but this does not explain the inferior activation of the 4-1BBL-GFP transduced population. Given that 4-1BB expression has been reported to suppress 4-1BBL expression, we postulated that expression of 4-1BBL reciprocally suppresses 4-1BB expression on the same cell, thus rendering the transduced population less responsive to 4-1BBL in trans.

Membrane expression of 4-1BBL down-regulates 4-1BB at the cell surface

To examine the relationship between constitutive expression of 4-1BBL and 4-1BB surface expression, bone-marrow derived murine DC were transduced with increasing quantities of 4-1BBL-GFP and expression of the ligand and receptor was analysed 2 days later. Around 40% of untransduced mouse DC at day 6 post-maturation expressed 4-1BB. This 4-1BB+ve group were equally susceptible to transduction as 4-1BB-ve (Figure 7A) as shown by an equivalent proportion of GFP positive DC. However, following transduction with 4-1BBL-GFP, 4-1BBL/4-1BB double positive cells were not observed (Figure 7A). As 4-1BBL expression was increased by addition of increasing amounts of 4-1BBL-GFP LV, 4-1BB surface expression was suppressed, however intracellular 4-1BB showed only a modest decline (Figure 7B). These data suggest restriction of 4-1BB surface expression by 4-1BBL. As this was also observed with 4-1BBLTc (Figure 7B), interaction between 4-1BBL and 4-1BB leading to intracellular retention must occur between the extracellular or transmembrane domains of ligand and receptor. This control of 4-1BB surface expression by 4-1BBL expression provides a simple mechanism to explain bystander DC activation by 4-1BBL expressing DC.

Figure 7. 4-1BBL down-regulates surface expression of 4-1BB.

Figure 7

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(A) both 4-1BB+ve and −ve dendritic cells are susceptible to transduction by 4-1BBL-GFP lentiviral vector as demonstrated by GFP expression, but 4-1BB expression is down-regulated in the 4-1BBL expressing cells (B) 4-1BBL-GFP lentiviral vector, but not Null-GFP lentiviral vector causes down-regulation of surface (EC) but not intracellular (IC) 4-1BB expression.

41BBL transactivation of DC occurs in vivo

To test whether DC transactivation occurs in vivo, and could therefore explain the action in trans that we saw in vaccination (Figure 4) we injected groups of 4 mice in the flank with either 4-1BBL-GFP or Null-GFP and then collected draining inguinal lymph nodes. We then examined GFP negative DC in the draining LN at day 4 (Figure 8A); in these experiments less than 1% of cells in each case were GFP positive. This revealed both greater numbers of CD11c+ MHCII+ GFP −ve cells in the draining LN of mice receiving 4-1BBL vs Null-GFP (data not shown) and higher numbers of DC expressing CD80, CD86 or both (Figure 8A). Expression of 4-1BBL on a small number of DC thus appears to both recruit many additional DC to the draining LN and to activate them. To further characterise the degree of up-regulation of CD80 and CD86, mice were injected but LN pooled between groups of 4 mice and CD11c+ve cells isolated by bead separation. Cells were then stained for CD11c, MHC II and CD80 or CD86 (Figure 8B). These data provide a clear rationale for LV encoded 4-1BBL as an effective adjuvant when expressed in trans with antigen expressed from a second LV vector.

Figure 8. 4-1BBL activates bystander dendritic cells in vivo.

Figure 8

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Mice were immunised in the flank with the lentiviral vectors shown then after 4 days draining lymph nodes were harvested and GFP negative cells analysed. (A) CD11C+ve, MHCII, CD80 and CD86 expression were analysed in lymph nodes from individual mice. (B) Lymph nodes were pooled from 4 mice, then CD11C+ve cells were isolated using magnetic bead separation, then cells were stained for CD11C, MHCII and CD80 or CD86. Paired T test, *p=0.01-0.04, **p=0.001-0.009.

Discussion

We have previously demonstrated that a number of intracellular activators can improve LV stimulation of anti-tumour and anti-influenza immune responses[27, 28]. We selected 4-1BBL as novel adjuvant candidate in LV because of its well-documented role in stimulating CD8+ T cell effector and memory responses. Following subcutaneous LV immunisation, which targets DC, we indeed found superior CD8+ T cell responses against influenza NP when 4-1BBL was also expressed by the LV. Vaccination with 4-1BBL and NP also conferred greater protection against lethal A/PR/8/34 challenge than NP. This is in agreement with a previous study using intranasal immunisation with an AV co-expressing 4-1BBL and NP[22].

Using LV immunisation, we also showed that 4-1BBL and NP should be expressed in trans for optimal quality of CD8+ T cell response. We have demonstrated that this is because 4-1BBL expressed on DC following LV immunisation can activate neighbouring, bystander DC expressing antigen. This is in contrast to the expression of 4-1BBL with Gag in a DNA vaccine, where only the cis configuration is effective[20]. However, the immune responses to an HBsAg DNA vaccine are substantially enhanced when a plasmid expressing 4-1BBL in trans is co-injected[39]. Also, an AV expressing 4-1BBL is at least partially effective in bone marrow chimeric mice with 41BB expression restricted to haematopoietic cells other than T cells, suggesting it can act via 4-1BB stimulation on antigen presenting cells[22]. In fact, despite the widespread experimental use of 41BB stimulation to enhance T cell responses to tumours or pathogens, it remains unclear the degree to which these responses are due to 4-1BB expressed on T cells. One confounding factor is that 4-1BB deficient T cells do not behave normally even in the absence of 4-1BBL. They undergo hyper-proliferation and reduced cytokine secretion the reason for which is not clear, so studies in mice lacking 4-1BB on T cells are difficult to interpret [40]. Together, these data suggest that both direct stimulation of T cells and also activation of antigen presenting cells such as DC should be considered when incorporating 4-1BBL into immunisation strategies.

Clearly, as DC express 4-1BBL during an immune response[13], this activation of neighbouring DC expressing 4-1BB may play a physiological role. For example, CD8+ lymphoid resident DC can cross-present antigen transferred from incoming migratory DC. The means by which migratory DC, activated by inflammatory mediators at the site of infection, pass on this activation pattern to recipient LN DC is unknown. It has been suggested that this occurs indirectly through activation of LN CD4+ T cells by migratory DC and subsequent licensing of LN resident DC by T-helper cytokines such as IL-4. However, it is also possible that a direct DC-DC signalling occurs in this and other scenarios mediated by 4-1BBL stimulation of 4-1BB.

As activated DC can express both 4-1BB and 4-1BBL, the inter-relationship between receptor and ligand expression upon DC activation has been examined. LPS up-regulates 4-1BBL on DC from 4-1BB−/− deficient mice to a much greater degree than WT[41], showing that 4-1BB expression suppresses 4-1BBL expression. A modest increase in 4-1BB expression on stimulated DC from 4-1BBL−/− deficient mice is also seen[2]. In our experiments we have demonstrated that constitutive 4-1BBL expression on DC causes potent 4-1BB down-regulation from the cell surface. As this down-regulation also occurs with a mutant 4-1BBL lacking the cytoplasmic tail, we propose that interaction between the extracellular and/or transmembrane domains of ligand and receptor sequesters the receptor within the cell. This could also be the mechanism by which 4-1BB inhibits 4-1BBL expression. The tight reciprocal control of surface expression of 4-1BBL and 4-1BB on DC does indeed suggest that co-expression of the two molecules on the same cell is restricted, perhaps to prevent propagation of self-sustaining DC activation.

Footnotes

1

This work was supported by an MRC Clinical Training Fellowship awarded to DCM and a grant from the UCLH NIHR Biomedical Research Centre.

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