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. Author manuscript; available in PMC: 2013 May 13.
Published in final edited form as: Eur J Immunol. 2010 Dec 3;41(1):29–38. doi: 10.1002/eji.201040717

TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity

Shirdi E Letran 1, Seung-Joo Lee 1, Atif Shaikh 1, Satoshi Uematsu 2,3, Shizuo Akira 2,3, Stephen J McSorley 1
PMCID: PMC3652676  NIHMSID: NIHMS276303  PMID: 21182074

Summary

Innate immune activation via Toll-like Receptors (TLRs) induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class-II presentation of flagellin epitopes to CD4 T cells and is required for induction of robust flagellin-specific adaptive immune responses. Flagellin-specific CD4 T cells expanded poorly in TLR5-deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin-specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5-deficient mice developed robust flagellin-specific T cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4 T cell responses to flagellin, indicating that TLR5 enhances flagellin-specific CD4 T cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin-specific CD4 T cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88-independent function for dendritic cell TLR5 in enhancing presentation of peptides to flagellin-specific CD4 T cells.

Keywords: TLRs, CD4 T cells, Dendritic cells

Introduction

Toll-Like Receptors (TLRs) are germline-encoded proteins that allow rapid immune reactivity to harmful pathogens [1]. Each TLR can recognize a common microbial signature that is foreign to the eukaryotic host. For example, TLR3 signaling is activated in response to viral, double-stranded RNA [2], TLR4 recognizes bacterial cell wall lipopolysaccaride (LPS) [3], and TLR5 detects the presence of bacterial flagellins [4–6]. TLR stimulation causes activation of NFkB, the production of inflammatory mediators, and rapid initiation of an anti-microbial response [1]. The importance of TLR recognition has been demonstrated in animal and human studies where TLR signaling deficiency has been shown to increase or decrease susceptibility to infectious disease [7–10].

Flagellin is the main component of the bacterial flagella and is produced in large quantities by many flagellated bacteria [11]. Flagellin activation of TLR5 involves the physical interaction of cell-surface TLR5 with the D1 domain of monomeric flagellin, a 3-dimentional structure containing conserved residues from the amino and carboxy-terminus [6, 12, 13]. The conserved structure of bacterial flagellins across different species allows host TLR5 to detect infections caused by diverse flagellated bacteria including Listeria, Salmonella, Legionella, and Pseudomonas. As with most other TLR family members, TLR5 signaling induces Myd88-dependent activation of MAP kinases, NF-kappaB activation, inflammatory cytokine production, and increased expression of MHC and co-stimulatory molecules on antigen presenting cells [4, 5, 14–16]. Thus, bacterial flagellins represent one of many conserved microbial structures that are directly recognized by the innate immune system.

However, unlike most other TLR ligands, flagellins are proteins and therefore have the potential to be directly targeted by pathogen-specific T cells. Our laboratory and others have identified MHC class-II epitopes of Salmonella flagellin [17–19], making this protein the most thoroughly characterized target antigen of CD4 T cells in the murine typhoid model [20–22]. Indeed, flagellin-specific CD4 T cells comprise a sizable fraction of the total Salmonella-specific CD4 T cell response and flagellin is also a major target antigen of Salmonella-specific B cell responses [18, 23, 24]. Furthermore, bacterial flagellins are dominant target antigens in mouse and human inflammatory bowel disease [25], where immune reactivity to flagellins correlates with increasingly severe disease [26, 27]. Thus, bacterial flagellins are somewhat unusual TLR ligands with the potential for dual recognition by the innate and adaptive immune response.

Although innate and adaptive immune responses to flagellins are well documented, it remains unclear if these responses are intrinsically linked [21]. Mice lacking TLR5 have deficient innate immune responses to bacterial flagellins and/or flagellated microbes, clearly demonstrating that TLR5 recognition of flagellin is involved in innate activation [15, 16]. However, a requirement for TLR5 in the adaptive immune response to flagellated pathogens or purified bacterial flagellins has yet to be carefully examined. Our hypothesis for this particular study was that the presence of an innate receptor for bacterial flagellin could serve to enhance the adaptive response to flagellin. Indeed, our data demonstrate that TLR5-deficient mice are unable to generate CD4 T cell responses to flagellin immunization, but this deficiency is overcome by delivery of processed peptide. Interestingly, this function of TLR5 was independent of the adaptor molecule Myd88 but required expression of TLR5 by dendritic cells. Together, these data suggest that TLR5 performs a novel Myd88-independent function as an endocytic receptor by enhancing presentation of flagellin peptides to antigen-specific CD4 T cells.

Results

TLR5-deficient mice display reduced CD4 T cell and antibody responses to flagellin

Previous reports have documented that injection of TLR5-deficient mice with bacterial flagellin failed to induce IL-6 or IL-12p40 or activate splenic dendritic cells [15, 16], demonstrating that TLR5 expression is required for these innate inflammatory responses. However, the role played by TLR5 during the adaptive responses to a natural MHC class-II epitope of flagellin has yet to be examined.

We previously developed a TCR transgenic adoptive transfer system that allows direct visualization of flagellin-specific CD4 T cell activation and expansion [28], and decided to use this methodology to examine the T cell response to flagellin in TLR5-deficient mice. Flagellin-specific SM1 T cells were detected at similar frequency in the secondary lymphoid tissues of wild-type and TLR5-deficient mice (Fig. 1A, upper left). Injection of 1µg of purified flagellin to wild-type mice induced significant SM1 T cell expansion and CFSE-dye dilution at day 3, and allowed the detection of a contracted antigen-experienced SM1 population at day 10 (Fig. 1A). In marked contrast, SM1 T cells failed to expand significantly in TLR5-deficient mice, although some of these cells had completed at least one cell division at day 3 and day 10 after immunization (Fig. 1A and B). Indeed, a marked deficiency in clonal expansion of SM1 T cells was observed in TLR5-deficient mice even after immunization with 200µg of flagellin (Fig. 1C). These data demonstrate that TLR5-deficient mice also have a marked deficiency in the expansion of CD4 T cell responses after immunization with flagellin.

Figure 1. TLR5-deficient mice are unable to mount a CD4 T cell response to flagellin immunization.

Figure 1

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Wild-type and TLR5-deficient mice were adoptively transferred with 600,000 CFSE-labeled SM1 T cells and immunized intravenously the following day with 1µg of purified Salmonella flagellin. Three and ten days later, spleens were harvested and stained with antibodies specific for CD4 and CD90.1 to detect SM1 T cells. (A) Representative FACS plots with numbers showing the percentage of SM1 T cells within the CD4+CD90.1+ boxed gate, and CFSE staining of SM1 T cells in lower plots. (B) Graph shows the percentage of SM1 T cells in individual flagellin immunized Wild-type and TLR5-deficient mice with bars representing the mean percentage of SM1 T cells. This graph shows pooled results from three individual experiments. The mean percentage of SM1 T cells was significantly higher at day 3 in Wt compared to TLR5-deficient mice by student t-test, *** p<0.0001. (C) Representative FACS plots with numbers showing the percentage of SM1 T cells within the CD4+CD90.1+ boxed gate three days after immunization of Wild-type or TLR5-deficient mice with 200µg of purified flagellin.

In order to assess B cell responses, we examined the production of flagellin-specific IgG in immunized wild-type and TLR5-deficient mice. After a single injection of flagellin, wild-type mice developed a flagellin-specific IgG response that was detectable 14 and 28 days later (Fig. 2A). In contrast, the flagellin-specific IgG response in TLR5-deficient mice fell below the limit of detection at both of time points (Fig. 2A). To examine a secondary response, each group received a second injection of flagellin at day 28 and flagellin-specific IgG was examined at day 40. Wild-type mice developed a high titer of flagellin-specific IgG after boosting, while a low but detectable response was present in TLR5-deficient mice (Fig. 2B). Together, these data demonstrate that TLR5 is required for optimal T and B cell responses to flagellin immunization.

Figure 2. TLR5-deficient mice have deficient flagellin-specific antibody responses.

Figure 2

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Wild-type and TLR5-deficient mice were immunized intravenously with 50µg of purified Salmonella flagellin. (A) Fourteen and twenty-four days later, sera was collected and examined for the presence of flagellin-specific IgG by ELISA. Graph shows the titer of flagellin-specific IgG in individual mice and bars show the mean titer. Responses in TLR5-deficient mice were below the limit of detection (shown by dashed line) at every time point examined. (B) Mice received a second injection of 50µg of purified flagellin, sera was collected at day 40 (day 16 post-boosting), and flagellin-specific IgG determined by ELISA. Graph shows the titer of flagellin-specific IgG in individual mice and bars show the mean titer +/− SD. The mean IgG titer was significantly higher at day 40 in Wt compared to TLR5-deficient mice by student t-test, * p<0.05.

TLR agonists fail to restore flagellin-specific responses in TLR5-deficient mice

It seemed possible that the inability of TLR5-deficient mice to generate a robust adaptive immune response after flagellin immunization was due to the absence of an adjuvant, since these mice display reduced inflammatory responses to flagellin [15, 16]. In order to examine this contribution of inflammation, we immunized TLR5-deficient mice with flagellin plus additional TLR4 (LPS), TLR2 (Pam3CSK), or TLR9 agonists (CpG DNA), and monitored the expansion of flagellin-specific CD4 T cells. In Wild-type mice, the addition of TLR2, 4, or 9 agonists had little effect on the maximal clonal expansion of SM1 T cells at day 3, but did enhance the frequency of SM1 cells detected at day 10 (Fig. 3). In contrast, injection of TLR5-deficient mice with flagellin plus TLR2, 4, or 9 agonists caused a minimal increase in SM1 T cells at day 3, and did not affect flagellin-specific CD4 T cell frequencies at day 10 (Fig. 3).

Figure 3. TLR2 and TLR4 agonists fail to rescue flagellin-specific CD4 responses in TLR5-deficient mice.

Figure 3

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Wild-type and TLR5-deficient mice were adoptively transferred with 600,000 SM1 T cells and immunized intravenously the following day with 1µg of purified Salmonella flagellin with or without 5µg of LPS or Pam3CSK4. Three and ten days later, spleens were harvested and stained with antibodies specific for CD4 and CD90.1 to detect SM1 T cells. (A) Representative FACS plots at day 3 and 10 post immunization with numbers showing the percentage of SM1 T cells within the CD4+CD90.1+ boxed gate (B) Graph shows the percentage of SM1 T cells in individual immunized Wild-type and TLR5-deficient mice at day 3 post-immunization with bars representing the mean percentage of SM1 T cells. This graph shows pooled results from two individual experiments. The mean percentage of SM1 T cells was significantly higher in Wt immunized with flagellin, flagellin/LPS, flagellin/PAM3CSK, or flagellin/CpG compared to immunized TLR5-deficient mice as assessed by student t-test, *** p<0.0001, * p<0.05.

These data indicated that the reduced ability of TLR5-deficient mice to generate flagellin-specific T cell responses was not due to a deficiency in inflammatory responses. An alternative mechanism could be that TLR5-deficient mice are unable to process flagellin for class-II presentation. Before we examined this issue directly, it was important to determine whether TLR5-deficient mice could mount a normal class-II restricted immune response to an irrelevant antigen that does not bind TLR5. We therefore monitored expansion of ovalbumin (OVA)-specific OT-II CD4 T cells in wild-type and TLR5-deficient mice following immunization with OVA. In contrast to our findings with flagellin-specific CD4 T cells, OVA-specific CD4 T cells expanded similarly in both wild-type and TLR5-deficient mice when either LPS or Pam3CSK was used as an adjuvant (Fig. 4A and B). Therefore, TLR5-deficient mice are able to process antigens for class-II presentation and can mount effective CD4 responses to non-flagellar antigens.

Figure 4. CD4 T cells respond to immunization with OVA or flagellin peptide in TLR5-deficient mice.

Figure 4

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(A, B) Wild-type and TLR5-deficient mice were adoptively transferred with 1 million OT-II T cells and immunized intravenously the following day with 100µg of Ovalbumin and 10µg of LPS. Three and ten days later, spleens were harvested and stained with antibodies specific for CD4 and CD90.1 to detect OT-II T cells. (A) Representative FACS plots are shown for mice at day 3 post immunization with numbers showing the percentage of OT-II T cells within the CD4+CD90.1+ boxed gate. (B) Graph shows the percentage of OT-II T cells in individual immunized Wild-type and TLR5-deficient mice at day 3 and 10 post-immunization with bars representing the mean percentage of OT-II T cells. This graph shows pooled results from two individual experiments. (C) Wild-type and TLR5-deficient mice were adoptively transferred with 800,000 SM1 T cells and immunized intravenously the following day with 100µg of flagellin peptide (427–441) and 10µg of LPS. Three days later, spleens were harvested and stained with antibodies specific for CD4 and CD90.1 to detect SM1 T cells. Representative FACS plots are shown for mice at day 3 post immunization with numbers showing the percentage of SM1 T cells within the CD4+CD90.1+ boxed gate. These plots are representative of 3 individual mice per group and two independent experiments. Expansion of OT-II T cells in response to OVA, or SM1 T cells in response to peptide was not statistically different in Wt compared to TLR5-deficient mice.

TLR5-deficient mice display normal CD4 responses to processed flagellin

Given the data above, it seemed possible that TLR5 was required for the processing of flagellin peptides for presentation on MHC class-II molecules. The flagellin epitope that is recognized by SM1 T cell has previously been previously mapped to peptide 427–441 [18]. Therefore, we examined whether SM1 T cells could respond in TLR5-deficient mice if this peptide was used as an immunogen rather than whole protein. Indeed, SM1 T cells expansion and memory development was identical in Wild-type and TLR5-deficient mice when flagellin peptide rather than whole protein was used to immunize mice (Fig. 4C). Thus, TLR5-deficient mice are unable to mount a CD4 T cell response to intact flagellin but respond normally to processed peptide, indicating that TLR5 is required for the processing of bacterial flagellins for antigen presentation to CD4 T cells.

Flagellin-specific T cells expand in the absence of other TLRs or Myd88

Our data suggested a surprising requirement for TLR5 for the processing of flagellin peptides and for subsequent flagellin-specific CD4 T cell activation. Myd88 is the only known adaptor molecule required for innate immune signaling downstream of TLR5 ligation [4, 5]. It was therefore of interest to determine whether induction of flagellin-specific CD4 T cell responses required expression of Myd88. Wild type and Myd88-deficient mice were adoptively transferred with SM1 T cells, immunized with flagellin, and the expansion of flagellin-specific CD4 T cells examined. Unlike TLR5-deficient mice (Fig. 1 and 3), SM1 T cells expanded in Myd88-deficient mice immunized with flagellin, although the peak response was modestly reduced compared to wild type mice (Fig. 5A and B). However, if LPS was provided as an additional adjuvant, flagellin-specific T cells expanded robustly in Myd88 and wild type mice (Fig. 5A and B). These data indicate that TLR5 expression is essential for the expansion of flagellin-specific CD4 T cells but that this function of TLR5 is largely Myd88 independent. Given the difference between TLR5-, and Myd88-deficient mice, we also examined whether there was any deficiency in the expansion of flagellin-specific CD4 T cells in TLR2-, or TLR4-deficient mice. While SM1 T cells were unable to expand in TLR5-deficient mice, significant expansion was detected in both TLR2-, and TLR4-deficient mice (Fig. 5C). Thus, the inability of flagellin-specific T cells to expand is a unique aspect of TLR5-deficient mice and is not found in other TLR-deficient mice.

Figure 5. Flagellin specific T cell responses are generated in Myd88-deficient mice.

Figure 5

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Wild-type and, (A and B) Myd88-deficient mice or, (C) TLR5-, TLR2-, or TLR4-deficient mice were adoptively transferred with 1 million SM1 T cells and immunized intravenously the following day with 1µg of purified flagellin. Three days later, spleens were harvested and stained with antibodies specific for CD4 and CD90.1 to detect SM1 T cells. (A) Representative FACS plots are shown for mice at day 3 post immunization with numbers showing the percentage of SM1 T cells within the CD4+CD90.1+ boxed gate. (B) Plots shows the percentage of SM1 T cell in immunized wild type or Myd88-deficient mice. Expansion of SM1 T cells in response to flagellin was not statistically different in Wt compared to TLR5-deficient mice. (C) Representative FACS plots are shown for mice at day 3 post immunization with numbers showing the percentage of SM1 T cells within the CD4+CD90.1+ boxed gate. These plots are representative of 3 individual mice per group.

TLR5-deficient dendritic cells are unable to activate flagellin-specific CD4 T cell in vitro

Our data suggested a novel role for TLR5 in antigen presentation in vivo. However, it was unclear whether this effect required TLR5 expression by antigen presenting cells themselves, and the identity of the antigen-presenting cell involved was also undefined. We therefore attempted to recapitulate this system in vitro by using purified murine CD11c+ dendritic cells. As expected, dendritic cells from wild-type mice were able to process whole flagellin and activate SM1 T cells in vitro to increase expression of CD25 and CD69 within 16 hours (Fig. 6A). In marked contrast, TLR5-deficient dendritic cells failed to activate SM1 T cells in vitro (Fig. 6A), even if anti-CD28 was provided as additional costimulation (Fig. 6B). Similar to the in vivo data above, dendritic cells from Myd88-deficient mice were fully capable of activating SM1 T cells in vitro (Fig. 6C). In addition to the increased expression of CD25 and CD69, dendritic cells from wild-type and Myd88-deficient mice were also able to induce the production of IL-2 from SM1 T cells, while dendritic cells from TLR5-deficient mice were not (Fig. 6D). Together, these data demonstrate that dendritic cell expression of TLR5 is required for optimal presentation of flagellin epitopes and that this function of TLR5 is Myd88-independent.

Figure 6. Splenic dendritic cells require TLR5, but not Myd88, to activate flagellin specific T cell responses in vitro.

Figure 6

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CD11c+ dendritic cells were purified from wild-type, TLR5-deficient, and Myd88-deficient mice and incubated with SM1 T cells in the presence of purified flagellin, Ovalbumin, or flagellin peptide 427–441. Sixteen hours later cells were harvested and stained with antibodies specific for CD4, CD90.1 (to detect SM1 T cells), and CD69 and CD25, before data was acquired using a flow cytometer. (A) Representative CD69 and CD25 histograms are shown for gated SM1 T cells from cultures with wild type or TLR5-deficient dendritic cells and are representative of three wells per group and three independent experiments. (B) Representative CD69 histograms are shown for gated SM1 T cells from cultures with wild type or TLR5-deficient dendritic cells with and without anti-CD28 and are representative of three wells per group and three independent experiments. (C) Representative CD69 histograms are shown for gated SM1 T cells from cultures with wild type or Myd88-deficient dendritic cells and are representative of three wells per group and three independent experiments. (D) IL-2 was measured by ELISA from the supernatant of cultures described in (A). Data show mean IL-2 production +/− SD for each sample and are representative of three independent experiments.

Discussion

Innate receptors initiate a rapid inflammatory response to infection, allowing early mobilization of immune cells to the infected site and elaboration of local defense mechanisms to combat the invading pathogen [1]. During this early protective innate response, microbe-specific lymphocytes clonally expand until they reach a level where pathogen eradication occurs and immune memory to secondary challenge is established [29]. It has been well documented that TLR5 recognition of flagellin plays an important role the development of innate immune responses to infection [30–33]. However, the involvement of TLR5 in the generation of adaptive immune responses has not yet been examined in any detail.

A major finding of our study is that adaptive immune responses to bacterial flagellin fail to develop normally in TLR5-deficient mice. Interestingly, this deficiency was not simply due to the absence of sufficient inflammatory mediators since the addition of TLR2, 4, or 9 agonists failed to recover flagellin-specific CD4 T cell responses. Instead, flagellin-specific T cell responses were completely recoverable by immunization with the major I-Ab-restricted peptide recognized by flagellin-specific CD4 T cells. Thus, our data point to a surprising requirement for TLR5 in enhanced processing of flagellin and suggest that TLR5 therefore functions as a scavenger receptor to enhance the delivery of flagellin to the class-II presentation pathway. Importantly, our data do not suggest that TLR5-deficient mice cannot make adaptive responses to flagellin, but simply that the access of flagellin to the class-II pathway is severely diminished to level of a nominal antigen such as OVA. It is also interesting that TLR5 enhanced CD4 T cell responses to flagellin epitopes while other studies have not observed the same effect when examining OVA fused to flagellin [34]. Greater study of endogenous and fused flagellin epitopes will be required in order to determine whether these antigens access the same processing pathway in vivo.

It should be noted that previous studies have clearly demonstrated that the inflammatory and adjuvant capacity of flagellin are dependent on Myd88 [31, 35–37]. However, since flagellin-specific CD4 responses were elicited in immunized Myd88-deficient mice, but not in TLR5-deficient mice, our data indicate that this particular role for TLR5 in scavenging flagellin for the class-II pathway is independent of Myd88 signaling. It seems possible that the enhancement of flagellin processing and/or presentation by TLR5 could occur simply a consequence of increased antigen uptake by virtue of a specific surface receptor or it may require signaling via an alternative adaptor pathway. As no alternative signaling pathway has yet been detected for TLR5, we favor the former hypothesis. The fact that this system could be replicated in vitro using purified CD11c+ dendritic cells strongly suggests that TLR5 expression by antigen presenting cells themselves is required for enhanced presentation of flagellin eptitopes. Our ability to demonstrate a TLR5-specific effect using splenic dendritic cells in vitro is somewhat surprising since previous reports have indicated that these cells do not express TLR5 [15]. However, our data clearly demonstrate that these dendritic cells are able to enhance presentation in a TLR5-dependent manner, even if they do not produce a marked inflammatory response to flagellin stimulation. Further analysis of splenic dendritic cell expression of TLR5 is required, especially since the TLR5-dependent effect on presentation to CD4 T cells appears to be a very sensitive assay in vitro.

Our data are interesting because they demonstrate a direct link between the specificity of an innate receptor and the induction of an adaptive response to the same ligand. It is currently unclear whether this might be a general feature of innate immune receptors that happen to be expressed by antigen presenting cells or whether this will be restricted to a subset of unusual protein ligands like flagellin. Indeed, a previous study has documented a link between TLR11 ligation by toxoplasma profilin and the immunodominance of profilin-specific CD4 T cells in this infection model [38]. Thus, enhancement of antigen presentation subsequent to TLR ligation is likely to be a common feature in infectious disease models and may allow rapid focusing of the adaptive response to major antigenic determinants such as profilin or flagellin, or to protein antigens that happen to be physically coupled to non-protein TLR ligands. Similarly, these data help explain why flagellin is a dominant target antigen in some models of inflammatory bowel disease [25–27], although a role for TLR5 in the generation of these responses has not yet been established.

In addition to CD4 T cell responses, our data also indicate a major deficiency in the induction of flagellin-specific IgG responses in TLR5-deficient mice. A recently published study concluded that TLR5-deficient mice had no deficiency in the induction of flagellin-specific IgG responses [39], while our data report a significant deficiency under almost identical experimental conditions. TLR5-deficient mice have also been reported to experience rectal prolapse or a metabolic syndrome [40, 41], yet neither of these phenotypes are present in our TLR5-deficient colony (data not shown). It is therefore likely that the presence of a co-factor derived from microflora is responsible for basal inflammatory disease phenotype in some animal colonies and that this inflammation masks detection of the requirement for TLR5 in induction of antibody responses to flagellin.

A role for TLRs in the selection of antigens for class-II presentation has previously been highlighted [42]. In this case, association of antigen and TLR ligands in the same particle was required for the generation of an optimal adaptive immune response [43], since this allows TLR signaling to occur within the same endosome where antigen is present. Obviously, in the situation of flagellin, where the TLR ligand is a protein and also a CD4 target, no artifical association of antigen and adjuvant is required. However, our data do not fit well within this model since in our experiments flagellin-specific T cell responses were detected in Myd88-deficient mice. This demonstrates that the innate inflammatory activity of flagellin can be uncoupled from the role of TLR5 in antigen processing. Thus, this model may require some adjustment to account for enhanced antigen processing that can be driven directly by innate receptor targeting of a ligand to the class-II pathway. Future experiments are planned in our laboratory to examine the mechanism of Myd88-independent TLR enhancement of flagellin antigen processing.

In conclusion, we report the finding that TLR5 essentially functions as an endocytic receptor for enhancing flagellin antigen processing to CD4 T cells. This function of TLR5 is independent of the only known adaptor molecule for TLR5 signaling, Myd88, and therefore uncovers a novel pathway where TLR5 can contribute to adaptive immunity. The reduction in flagellin targeting in TLR5-deficient mice also provides a mechanistic explanation for the targeting of bacterial flagellins in inflammatory and infectious diseases.

Materials and Methods

Mouse and bacterial strains

C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and the Jackson Laboratory (Bar Harbor, ME) and used at 6–12 weeks of age. Myd88- [44], and TLR5-deficient [15] mice were bred at the University of Minnesota from breeding stock originally provided by Dr. S. Way (University of Minnesota) and Dr. A. Gewirtz (Emory University). RAG-deficient SM1 and OT-II TCR transgenic mice expressing CD90.1 or CD45.1 alleles have previously been described [28, 45–47], and these lines were intercrossed and screened in our mouse colony. TLR2-, and TLR4-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). LPS-deficient S. typhimurium X4700 was generously provided by Dr. R. Curtiss, Arizona State University, AZ.

Immunization of mice

Flagellin was purified from S. typhimurium (X4700) using a modified acid-shock protocol [31, 48]. Bacteria were grown at 37°C without shaking, before being washed and re-suspended in PBS/HCl (pH 2) for 30 minutes at room temperature. Bacterial supernatants were collected and ultracentrifugation and ammonium sulphate precipitation was used to purify flagellin. Residual LPS was removed by serial passage through multiple detoxigel columns (Pierce Biotechnology). Silver-stained SDS-PAGE gels were used to confirm purity of flagellin preparations and each batch of flagellin was found to be LPS-free using the Limulus assay. Mice were immunized intravenously with 1–200µg Salmonella flagellin, 200µg ovalbumin, or 100µg flagellin peptide (427–441) with or without the addition of 5µg of LPS (Sigma), CpG DNA (Midland Certified Reagent Company, Midland, TX), or Pam3CSK4 (InvivoGen, San Diego, CA and Alexis Biochemicals, San Diego, CA). Various times later, mice were sacrificed to obtain serum and spleens for flow cytometry analysis. In pilot experiments mice were immunized with several different doses of bacterial flagellin and we determined that 1µg was sufficient to generate maximal CD4 T cell expansion in vivo (data not shown). This dose was therefore used for most experiments and is in the same range previously used to examine the specific adjuvant effect of flagellin via TLR5 [34].

Analysis of antibody responses

Blood was collected retro-orbitally at various time points from immunized mice and sera prepared by centrifugation. High protein binding plates were coated with flagellin or heat-killed S. typhimurium diluted in 0.1M NaHCO3 and incubated overnight at 4°C. After incubation in Fc block (spent culture supernatant from the 24G2 hybridoma, 2% rat serum, 2% mouse serum, and 0.01% sodium azide) for one hour at 37°C, plates were washed twice in PBS/0.05% Tween 20. Samples were added in serial dilutions, diluted in 10% FCS/PBS, and incubated for two hours at 37°C. Plates were washed four times before biotin-conjugated antibody specific for the desired isotype was added. After incubation for one hour at 37°C, plates were washed six times. Finally, plates were incubated for one hour at 37°C in alkaline phosphatase diluted in 10% FCS/PBS. Plates were washed eight times and a substrate containing sodium phosphate, citric acid, O-phenylenediamene, and H2O2 was added. After sufficient color-change was observed, 2N H2SO4 was added to stop the reaction before plates were analyzed using a spectrophotometer.

TCR transgenic adoptive transfers and analysis

Spleen and lymph node cells (inguinal, axillary, brachial, cervical, mesenteric, and peri-aortic) were harvested from SM1 or OT-II mice and a single cell suspension was generated. An aliquot of this sample was stained using antibodies to CD4 and the relevant TCR Vβ in order to determine the percentage of TCR transgenic cells. Volumes were adjusted accordingly and 1×105–1×106 SM1 or OT-II cells were injected intravenously into recipient C57BL/6 mice. In most experiments the TCR transgenic cells were stained with CFSE [49] immediately prior to adoptive transfer. At various time points after immunization, spleens were harvested and a single cell suspension generated in complete EHAA medium. Samples were incubated on ice for 30 minutes in Fc block containing FITC-, PE-, PE-Cy5-, or APC-conjugated antibodies specific for CD4, CD11a, CD25, CD69, CD45.1, or CD90.1 (eBioscience and BD Bioscience). After staining, cells were fixed using paraformaldehyde and examined by flow cytometry using a FACS Canto. All flow data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

Purification of dendritic cells and in vitro assays

Spleens were harvested from mice and incubated with collagenase D (37°C for 20 minutes) and EDTA to liberate dendritic cells, as previously described [31]. Magnetic anti-CD11c microbeads and multiple passes through selection columns (Miltenyi Biotech, Auburn, CA) were used to isolate CD11c+ spleen dendritic cells to 85–95% purity. Purified dendritic cells (1×105/well) were washed and placed in culture with SM1 T cells (1×105/well) plus titrated concentrations of flagellin protein, flagellin peptide (427–441), or ovalbumin and cultured for 16 hours in 96-well tissue culture plates. In some cultures anti-CD28 (5µg/ml) was added to provide additional costimulation. Individual wells were harvested and stained for antibodies specific to CD4 and CD90.1 (to detect SM1 T cells) and surface activation molecules CD25 and CD69. Samples were acquired using a FACS Canto flow cytometer and data analyzed using FlowJo. Supernatants from in vitro cultures were collected and stored at −80°C before the concentration of IL-2 was determined by sandwich ELISA.

Statistical analysis

Data were first determined to be normally distributed and differences between groups examined using InStat (GraphPad Software, La Jolla, CA). Data in each group were compared using an unpaired t test and were considered significantly different with a p value of <0.05.

Acknowledgements

This work was supported by grants from the National Institutes of Health AI073672 and AI055743.

Footnotes

Conflict of Interest

The authors have received no financial or commercial benefits associated with these data and declare that no conflict of interest exists.

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