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The Journal of Immunology, 2008, 181, 2392 -2398
Copyright © 2008 by The American Association of Immunologists, Inc.
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Activation of {gamma}{delta} T Cells by Borrelia burgdorferi Is Indirect via a TLR- and Caspase-Dependent Pathway1

Cheryl Collins, Cuixia Shi, Jennifer Q. Russell, Karen A. Fortner and Ralph C. Budd2

Immunobiology Program, Department of Medicine, The University of Vermont College of Medicine, Burlington, VT 50405


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are {gamma}{delta} T cells. {gamma}{delta} T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human {gamma}{delta} T cells of the V{delta}1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine {gamma}{delta} T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of {gamma}{delta} T cells, are both caspase-dependent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lyme disease is the most common vector-borne disease in the United States (1, 2), which is caused by the spirochete Borrelia burgdorferi and transmitted to humans by Ixodes ticks (3, 4). Infection often produces a distinct rash and flu-like illness in its initial stage, but can later progress to more chronic cardiac, neurologic, and rheumatic manifestations (1, 5, 6, 7). Several lines of evidence point to a role of T lymphocytes in Lyme arthritis. These include the ability of adoptively transferred B. burgdorferi-specific T cells to confer arthritis (8), the presence of activated T cells in Lyme arthritis synovial tissue that proliferate to B. burgdorferi (7, 9), an association of HLA-DR4 with chronic antibiotic-resistant Lyme arthritis (10), similar to rheumatoid arthritis (11) and effective therapeutic treatments that partially inhibit T cell function (1, 2).

{gamma}{delta} T cells are activated in a variety of infectious and inflammatory disorders. They are often anatomically sequestered at epithelial barriers or sites of inflammation (12), and can manifest cytotoxicity toward a wide array of targets (13). {gamma}{delta} T cells are moderately protective in infections due to Listeria (14), Leishmania (15), Mycobacterium (16), Plasmodium (17), and Salmonella (18). {gamma}{delta} T cells also accumulate at inflamed sites in autoimmune disorders such as rheumatoid arthritis (19), Lyme arthritis (20), celiac disease (21), and sarcoidosis (22). Evidence suggests that they may be beneficial in certain autoimmune models. Both collagen-induced arthritis in mice (23) and adjuvant arthritis in rats (24) are worse after depletion of {gamma}{delta} T cells, as are murine lupus (25) and a model of orchitis (26).

The predominant human {gamma}{delta} T cell subset in peripheral blood is V{gamma}2V{delta}2, which reacts to nonpeptide Ags from Mycobacterium. These include isoprenyl pyrophosphates (27, 28, 29, 30), as well as alkylamine Ags (31). These are products of microbes as well as self-Ags. By contrast, the V{delta}1 subset accumulates in inflamed synovium in rheumatoid (19, 32) and Lyme arthritis (20). Very little is known regarding the mechanism of activation of this {gamma}{delta} T cell subset or their function. Our earlier studies on synovial V{delta}1 cells revealed that they can potently activate myeloid dendritic cells (DC)3 in a Fas/Fas-ligand-dependent manner (33). In this capacity, V{delta}1 cells may be important at initiating the adaptive immune response. We have also previously observed that Lyme synovial V{delta}1 cells proliferate in response to a sonicate of Borrelia burgdorferi plus DC, but it was uncertain whether this process was a direct or indirect activation by Borrelia components. We now observe that activation of V{delta}1 cells by B. burgdorferi is predominantly an indirect process via TLR signaling and requires cell contact with metabolically active DC or monocytes. Studies with both human V{delta}1 T cells and murine {gamma}{delta} T cells show that Borrelia activates {gamma}{delta} T cells primarily via TLR2, but stimulation by other TLR ligands can also indirectly activate {gamma}{delta} T cells. These new findings serve to form a stronger connection by {gamma}{delta} T cells between innate and adaptive immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice, either wild-type, TLR2–/–, TLR9–/–, or MyD88–/–, were housed and bred in the University of Vermont animal facility and used at 2–6 mo of age. The facility is American Association of Laboratory Animal Care approved, and protocols were approved by Institutional Animal Care and Use Committee. Original breeding pairs were obtained from The Jackson Laboratory.

Derivation of synovial fluid {gamma}{delta} T cell clones and monocyte-derived DC

Lymphocytes were purified from synovial fluid of Lyme arthritis patients by Ficoll-Hypaque centrifugation (Sigma-Aldrich), and cultured in AIM-V medium (Life Technologies) containing 5% FBS (HyClone) and 50 U/ml recombinant human IL-2 (Cetus). Cells were stimulated with 10 µg/ml of a sonicate of B. burgdorferi, strain N40, grown in BSK II medium (Sigma-Aldrich) as previously described (20, 34). From these bulk cultures, responding cells were cloned at 0.3 cells/well in AIM-V with 10% FBS in the presence of irradiated peripheral blood lymphocytes (3 x 105/well), human rIL-2 (100 U/ml), and 10 µg/ml B. burgdorferi. After 14–21 days, cells from positive wells were phenotyped and those containing {gamma}{delta}+ T cells were expanded by restimulation with either B. burgdorferi (10 µg/ml) or PHA (1 µg/ml) (Murex Biotech), at ~14-day intervals. All synovial {gamma}{delta} clones were V{delta}1 by Ab screening and DNA sequencing and proliferated in response to Borrelia stimulation (35).

Human monocytes were purified as CD14+ cells from peripheral blood of healthy volunteers (Miltenyi Biotec). Myeloid DC were prepared by culture of monocytes with 800 U/ml GM-CSF, (BioSource International) and 500 U/ml IL-4 (BioSource).

Preparation of murine {gamma}{delta} T cells and bone marrow-derived dendritic cells (BMDC)

Spleen cells were depleted of erythrocytes by hypotonic lysis followed by negative selection to enrich for {gamma}{delta} T cells using rat mAbs to CD4 (GK1.5), CD8 (Tib105), B220 (RA3–6B2), MHC class II (3F12), and CD11b (M1/70) for 30 min. The samples were washed and then incubated with goat anti-rat IgG-labeled magnetic beads (Qiagen) for 45 min followed by magnetic field separation. The purified cells were cultured in 48-well plates coated with 5 µg/ml anti-TCR-{gamma}{delta} Ab (GL3) in complete culture medium (RPMI 1640 supplemented with 25 mM HEPES, 2.5 mg/ml glucose (Sigma-Aldrich), 10 µg/ml folate (Invitrogen Life Technologies), 110 µg/ml pyruvate (Invitrogen Life Technologies), 5 x 10–5 M 2-ME (Sigma-Aldrich), 292.3 µg/ml glutamine (Invitrogen Life Technologies), 100 U/ml penicillin-streptomycin (Life Technologies), and 10% FBS) containing 100 U/ml recombinant human IL-2. After 2 days, cells were moved to uncoated wells for further expansion with complete medium plus IL-2. On day 7, cells were used for experiments with {gamma}{delta} T cell purity >95%.

The preparation of BMDC was done according to the method of Lutz et al. (36) and used on day 10.

T cell-monocyte/DC cocultures

Cultures of monocytes or DC (5 x 105/ml) with V{delta}1 clone cells (1 x 106/ml) were made in AIM-V medium with IL-2 (100 U/ml) and 10% FBS in the absence or presence of B. burgdorferi sonicate (10 µg/ml), purified native lipidated or delipidated OspA from B. burgdorferi as previously described (35) (10 µg/ml), or in some experiments other TLR ligands, including Pam3Cys (InvivoGen), E. Coli 0111:B4LPS (Sigma-Aldrich), CpG (Coley Pharmaceutical), or Poly(I:C) (Sigma-Aldrich) (each at 1 µg/ml). After 24 h, cell supernatants were removed for cytokine analysis by ELISA, and cells were stained for expression of TCR-{gamma}{delta} and CD25 by flow cytometry using an LSR II (BD Biosciences). Blocking studies were performed using either control IgG, anti-TLR2 (a gift of Dr. Robert Finberg, University of Massachusetts, Worcester, MA), anti-IL-1β (clone 8516, R&D Systems), anti-IL-6 (cat. AF206NA, R&D Systems), and anti-TNF-{alpha} (clone J1D9, Ancell) (each at 20 µg/ml).

Transwell cultures were performed using 1 x 106 V{delta}1 cells in 1 ml AIM-V/FBS/IL-2 medium placed in the lower chamber, with 5 x 105 DC in 100 µl placed on top of the membrane of the upper chamber with B. burgdorferi sonicate at 10 µg/ml. After 24 h, the V{delta}1 cells were assessed for expression of CD25.

Chemical fixation of DC was performed by incubating the cells in the absence or presence of B. burgdorferi at 10 µg/ml at 37°C overnight, then washed in 5% FBS in RPMI 1640, and fixed by the addition of ice-cold 1-ethyl-3(3'-dimethyl-aminopropyl)-carbodiimide (Sigma-Aldrich) at 75 mM in PBS for 60 min on ice. Following fixation the DC were extensively washed with 5% FBS/RPMI 1640 and then incubated with V{delta}1 cells in the absence of additional B. burgdorferi. Expression of CD25 by the V{delta}1 cells was examined after an additional 24 h.

Abs and flow cytometry

Abs used were to the following determinants: TCR-{gamma}{delta} (5A6.E9, Invitrogen/Caltag), CD25 (CD25–3G10, Invitrogen/Caltag), CD1a (HI149, Invitrogen/Caltag), CD1b (M-T101, BD-Pharmingen), CD1c (M241 Ancell), and CD1d (CD1d42, BD Pharmingen). Samples were analyzed on an LSR II flow cytometer (BD Biosciences).

Biotin-VAD-fmk active caspase precipitation assay

Cells were lysed in lysis buffer containing 20 µM biotin-VAD-fmk (MP Biomedicals). In brief, 600 µg of lysate were precleared by rocking with 40 µl Sepharose 6B agarose beads (Sigma-Aldrich) at 4°C for 2 h. Supernatants were then rocked with 60 µl streptavidin-Sepharose beads (Zymed, Invitrogen) at 4°C overnight. Beads were washed five times in lysis buffer, then boiled in loading buffer. Beads were removed by centrifugation and immunoblot analysis was then performed on supernatants.

Immunoblot analysis

T cells were lysed in buffer containing 0.2% NP40, 20 mM Tris-HCl (pH 7.4, American Bioanalytical), 2 mM sodium orthovanadate (Sigma-Aldrich), 10% glycerol (Fisher Scientific), 150 mM NaCl (Sigma-Aldrich), complete protease inhibitor (Roche Diagnostics), and 20 µM z-VAD-fmk (MP Biomedicals). Protein concentration was determined by Bradford assay (Bio-Rad). Protein lysates were boiled for 5 min in loading buffer containing 2-ME and separated using SDS-PAGE on 10 or 12.5% gels. Proteins were transferred onto PVDF membranes (Bio-Rad) and blocked using 4% milk in Tris-buffered saline plus 0.1% Tween 20 (American Bioanalytical) at room temperature for 1 h. Membranes were incubated at 4°C overnight in milk containing anti-human caspase-8 (BD Biosciences). Immunoreactive proteins were visualized using HRP-labeled conjugates (Santa Cruz Biotechnology; Southern Biotech; Biomeda) and developed using LumiGlo (KPL).

Caspase activity assay

Relative caspase activities were determined using the Apo-ONE Caspase Assay (Promega). Monocytes, either freshly isolated or following 2 day culture with B. burgdorferi (10 µg/ml) or cultured DC were resuspended in culture medium at 10 x 106 cells/ml. One hundred microliters of cells were serially diluted in 100 µl culture medium and then mixed with 100 µl of caspase reagent (DEVD-rhodamine) according to the manufacturer’s protocol. Spectrophotometric readings were taken over a range of times using a fluorescence reader (Bioteck Instruments).

Statistical analysis

Unpaired t tests were used to assess the significance of differences in production of cytokinies by monocytes and DC assessed by ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
B. burgdorferi activates {gamma}{delta} T cells indirectly via TLR pathways

To examine the pathway by which B. burgdorferi might activate {gamma}{delta} T cells, we considered whether this would be a direct or indirect process. As shown in Fig. 1A, Lyme arthritis synovial V{delta}1 clones (20, 35), as represented by clone Bb03, were stimulated to express CD25 by the addition of B. burgdorferi in the presence of myeloid DC. Borrelia alone did not induce CD25 expression, and myeloid DC without Borrelia induced only a modest up-regulation of CD25 expression by the V{delta}1 clones. Borrelia stimulation of the V{delta}1 clones was also possible using fresh CD14+ monocytes from which the DC were derived, using HLA mismatched donors (Fig. 1B, lower panels). By contrast, activation of a Borrelia-specific CD4+ {alpha}β T cell clone, 114B, was achieved only by use of autologous monocytes (Fig. 1B, upper panels).


Figure 1
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  		FIGURE 1. Borrelia burgdorferi activates both human and murine {gamma}{delta} T cells. A, Induction of CD25 expression by human Lyme arthritis synovial V{delta}1 T cell clone Bb03 following 24 h of stimulation with medium alone (Bb03), a sonicate of B. burgdorferi alone (Bb03+Bb), DC alone (Bb03+DC), DC plus B. burgdorferi (Bb03+DC+Bb), or fresh monocytes plus B. burgdorferi (Bb03+monos+Bb). The findings were consistent in three separate experiments using two V{delta}1 clones from two patients. B, Human CD4+ TCR-{alpha}β (h{alpha}β) Borrelia-specific T cell clone 114B (upper panel) was stimulated with monocytes and B. burgdorferi using either autologous monocytes (114B+monos 1+Bb) or HLA-mismatched monocytes (114B+monos 2+Bb). CD25 expression was measured after 24 h. A human V{delta}1 (hV{delta}1) clone Bb03 was used under identical stimulation conditions (lower panel). C, Murine splenic {gamma}{delta} T cells were purified by negative selection (see Materials and Methods) and then stimulated with anti-{gamma}{delta} plus IL-2 and propagated for 8 days until surface CD25 levels had down-regulated. The {gamma}{delta} T cells were then restimulated with syngeneic BMDC in the absence or presence of B. burgdorferi, and CD25 levels measured after 24 h. The findings were consistent in two experiments. D, Human V{delta}1 T cell clone Bb03 was stimulated with fresh CD14+ monocytes (monos) in the presence of either no additives, B. burgdorferi sonicate, lipidated OspA (OspA), or delipidated OspA (delip OspA). CD25 expression was measured after 24 h by flow cytometry and results expressed as the percent increase compared with minimum defined by {gamma}{delta} T cells with monos or DC alone, and maximum CD25 induction defined by stimulation with Borrelia sonicate plus DC. E, The same conditions as in C were used to activate murine {gamma}{delta} T cells with BMDC.

 
To further test the generality of the activation of {gamma}{delta} T cells by Borrelia, we also examined the activation of murine {gamma}{delta} T cells in this system. Splenic {gamma}{delta} T cells were enriched by negative selection and then activated with anti-{gamma}{delta} Ab plus IL-2 and expanded over 7 days. Cultures at this time contained >95% {gamma}{delta} T cells bearing low levels of surface CD25. As such, they resembled the activation state of the human synovial V{delta}1 T cell clones. The murine {gamma}{delta} T cells were then restimulated using syngeneic BMDC with or without B. burgdorferi. Similar to the human synovial V{delta}1 cells, murine splenic {gamma}{delta} T cells were also strongly stimulated to up-regulate CD25 in the presence of BMDC plus Borrelia, but to a much lesser extent with BMDC alone (Fig. 1C). Further dissection of the components of B. burgdorferi that were responsible for the stimulation of CD25 expression by the {gamma}{delta} T cells revealed that a single surface lipoprotein, OspA, was capable of stimulating both human V{delta}1 T cells (Fig. 1D) and murine {gamma}{delta} T cells (Fig. 1E) to nearly the same degree as the Borrelia sonicate. However, when the tripalmitic acid lipid component of OspA was absent, the ability to stimulate human and murine {gamma}{delta} T cells was lost.

Lipidated OspA has been shown to be a TLR2 ligand, and B. burgdorferi contains no LPS, a TLR4 ligand (37). We thus examined the requirement of TLR2 signaling in the activation of the human and murine {gamma}{delta} T cells. A blocking antihuman TLR2 Ab inhibited the induction of CD25 expression by the human V{delta}1 cells by ~50%, which was consistent in three experiments (Fig. 2A). A similar decline in CD25 induction was observed in the murine {gamma}{delta} T cells when BMDC were used from TLR2–/– mice (Fig. 2B). Less decline of CD25 expression was observed in the absence of TLR9 (a receptor for bacterial DNA), but in the absence of MyD88, a signaling intermediate for most TLR, there was essentially complete inhibition of CD25 induction (Fig. 2B). The murine {gamma}{delta} T cell findings were consistent in three experiments.


Figure 2
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  		FIGURE 2. Activation of human and murine {gamma}{delta} T cells by B. burgdorferi requires TLR signals. A, Human V{delta}1 T cell clone Bb03 was either not stimulated, or activated by DC plus Bb in the presence of no Ab, control IgG, or anti-TLR2, both at 20 µg/ml. CD25 expression was measured by flow cytometry after 24 h. B, Murine {gamma}{delta} T cells were stimulated in the absence (white bars) or presence (black bars) of B. burgdorferi sonicate using DC from wild-type C57BL/6 mice (B6+/+), or B6 mice deficient for TLR2, TLR9, or MyD88. CD25 expression was measured by flow cytometry after 24 h. The findings were consistent in two separate experiments.

 
The ability of Borrelia lipopeptides to activate both human and murine {gamma}{delta} T cells raised the question whether this phenomenon might be a property of other TLR ligands. We therefore examined the ability of several TLR ligands to activate human and murine {gamma}{delta} T cells. As shown in Fig. 3, in the presence of DC, Pam3Cys (TLR2 ligand), poly(I:C) (TLR3 ligand), LPS (TLR4 ligand), or CpG (TLR9 ligand) were each able to activate human (Fig. 3A) and murine (Fig. 3B) {gamma}{delta} T cells. As with Borrelia stimulation, no induction of CD25 expression by the V{delta}1 clones was observed in the absence of DC, and we have not observed expression of these TLR by the {gamma}{delta} T cells (data not shown). Collectively these findings suggested that activation of {gamma}{delta} T cells by B. burgdorferi and other TLR ligands is an indirect process.


Figure 3
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  		FIGURE 3. Ligands for various TLR can activate {gamma}{delta} T cells. A, Human V{delta}1 T cell clone Bb03 (A) or murine {gamma}{delta} T cells (B) were either not activated, or activated in the presence of DC with B. burgdorferi (Bb) or TLR ligands Pam3Cys (TLR2), Poly(I:C) (TLR 3), LPS (TLR4), or CpG (TLR9), and surface CD25 expression measured after 24 h. Results were consistent in three separate experiments.

 
Activation of human V{delta}1 cells by B. burgdorferi requires cell contact with DC

To determine whether cell contact between {gamma}{delta} T cells and DC was required for activation of {gamma}{delta} T cells, a transwell system was used in which V{delta}1 T cells were placed in the lower chamber (106/ml) and DC (5 x 105 in 100 µl) placed on the membrane of the upper chamber with or without B. burgdorferi. Cell densities and Borrelia sonicate concentration (10 µg/ml) were the same as used in conventional contact cultures, which were used as a positive reference control for induction of CD25 on {gamma}{delta} T cells. Fig. 4A shows that no induction of CD25 expression on human {gamma}{delta} T cells was observed in the transwell system using DC alone, and this was not significantly increased with the addition of Borrelia to the transwell cultures (Fig. 4A). To examine this further, supernatants from various DC culture conditions were used to activate human V{delta}1 cells. Neither supernatants from cultures of DC alone, nor DC plus Borrelia for 24 h could activate the {gamma}{delta} T cells (Fig. 4B). We further studied this question following fixation with 1-ethyl-3(3'-dimethyl-aminopropyl)-carbodiimide of human or mouse DC after 24 h incubation with Borrelia sonicate. Fixation resulted in the complete loss of the ability of DC to activate either human (Fig. 4C) or mouse (Fig. 4D) {gamma}{delta} T cells. However, DC pretreated with Borrelia in a similar manner, washed thoroughly after 24 h, but not fixed, were able to strongly activate human and mouse {gamma}{delta} T cells (Fig. 4, C and D). These collective findings supported the view that full activation of {gamma}{delta} T cells by B. burgdorferi required direct cell contact with metabolically active DC.


Figure 4
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  		FIGURE 4. Activation of {gamma}{delta} T cells requires contact with metabolically active DC. A, Transwell culture in which V{delta}1 clone Bb03 (106/ml) was placed in the lower chamber and DC (5 x 105 in 100 µl) placed on the membrane of the upper chamber, in the absence (Bb03/(DC)) or presence of B. burgdorferi sonicate (Bb03/(DC+Bb)). Bb03+DC+Bb served as a comparison for activation by conventional non-transwell contact stimulation, shown at the bottom. Surface CD25 was examined after 24 h by flow cytometry. B, V{delta}1 clone Bb03 was stimulated with supernatants (SN) from 24 h cultures of DC alone ((DC)SN) or DC + B. burgdorferi ((DC+Bb)SN). CD25 expression was measured after 24 h. Comparison was made to conventional stimulation of Bb03+DC+Bb shown at the bottom. Human (C) or murine (D) DC were incubated overnight with B. burgdorferi extract and then either fixed with 1-ethyl-3(3'-dimethyl-aminopropyl)-carbodiimide or unfixed before the addition of either human V{delta}1 T cell clone Bb03 (C) or murine {gamma}{delta} T cells (D) for an additional 24 h. CD25 expression was then measured gated on the {gamma}{delta} T cells. Findings are representative of three separate experiments.

 
To further examine whether secreted cytokines from APC might contribute to the activation of {gamma}{delta} T cells, we performed bioplex analysis of supernatants of monocytes and DC following stimulation for 24 h with B. burgdorferi. Findings for the most prominently secreted cytokines, IL-1β, IL-6, and TNF-{alpha}, were confirmed by ELISA, and are shown for monocytes in Fig. 5, A–C. Similar findings were observed for DC, with the exception of IL-1β (Fig. 5, A–C). Using this information, we attempted to use this cytokine combination to supplant the effects of DC alone or DC plus Borrelia in activating {gamma}{delta} T cells. However, synovial V{delta}1 clones cultured with a mixture of IL-1β plus IL-6 plus TNF-{alpha}, either in the absence or presence of DC, failed to up-regulate CD25 expression (Fig. 5D). Conversely, a combination of blocking Abs to these same cytokines did not diminish the induction of CD25 expression with DC plus Borrelia in conventional cultures (Fig. 5D). These results suggest that human V{delta}1 T cells respond to a surface determinant on Borrelia-activated DC and monocytes.


Figure 5
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  		FIGURE 5. Monocyte/DC-derived cytokines alone are insufficient to activate {gamma}{delta} T cells. Production of IL-1β (A), TNF-{alpha} (B), and IL-6 (C) by human monocytes (monos) or DC stimulated for 24 h with B. burgdorferi sonicate. Supernantants were analyzed by ELISA (*, p < 0.05 by unpaired t test). D, V{delta}1 clone Bb03 was cultured without or with a combination of IL-1β + IL-6 + TNF-{alpha} in the absence or presence of DC, and without B. burgdorferi. Alternatively, Bb03 was stimulated by DC plus Borrelia in the absence or presence of blocking Abs to IL-1β, IL-6, and TNF-{alpha}. CD25 expression was measured after 24 h. Findings were consistent in two experiments.

 
Caspase activity is required for activation of monocytes by B. burgdorferi and stimulation of CD25 by {gamma}{delta} T cells

The ligands for most human and murine {gamma}{delta} T cells are unknown, although suggestions have been made that certain human V{delta}1 T cells may react to CD1c (38) and that murine V{gamma}4 T cells may react to CD1d (39). We thus examined the expression of surface CD1 molecules on monocytes following Borrelia stimulation. As shown in Fig. 6A, surface expression by human monocytes of CD1a, CD1b, CD1c, and CD1d was up-regulated by 48 h of Borrelia stimulation. It was also recently reported that TLR activation of B cells required caspase activity (40). We therefore examined the effect of caspase blockade by the pan-caspase blocker z-VAD-fmk on CD1 expression and cytokine production by monocytes, as well as its effect on activation of the V{delta}1 T cells. Administration of vehicle control DMSO did not disturb the ability of B. burgdorferi to stimulate CD1 expression by monocytes. By contrast, the pan-caspase blocker z-VAD-fmk inhibited Borrelia induction of CD1 in a dose-dependent manner (Fig. 6, A and B). A similar induction of CD1d expression by Borrelia was also observed for murine DC that was also blocked by z-VAD (data not shown). Furthermore, z-VAD also blocked monocyte production of IL-1β and IL-12, but not IL-6 or TNF-{alpha} (Fig. 6C). This supports the view that z-VAD-fmk was not simply nonspecifically toxic to monocytes, but did block the up-regulation of several molecules, consistent with a role of caspases in TLR signaling by B. burgdorferi. Caspase activity was also critical for the ability of monocytes to activate synovial V{delta}1 clones. Fig. 6D shows that the presence of caspase blockade during the Borrelia stimulation of monocytes prevented the induction of CD25 by the {gamma}{delta} T cell clones.


Figure 6
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  		FIGURE 6. Caspase-dependent induction of expression of CD1 and certain cytokines by B. burgdorferi. A, Freshly isolated human CD14+ monocytes were cultured for 48 h in medium alone or B. burgdorferi plus vehicle control DMSO or the pan-caspase blocker z-VAD-fmk (100 µM). Monocytes were then stained for expression of CD1a, CD1b, CD1c, and CD1d. Similar results were observed in five experiments. B, Monocytes were stimulated with B. burgdorferi for 24 h in the absence or presence of DMSO or the indicated concentrations of z-VAD. Cells were then stained for expression of CD1b as well as with live/dead stain. Results are gated on live cells only and displayed as mean fluorescence intensity (MFI) of CD1b. C, Supernatants from the same cultures were assessed for levels of IL-1β, IL-12, IL-6, and TNF-{alpha} (*, p < 0.05 by unpaired t test). D, CD25 expression by V{delta}1 clone Bb03 after 24 h culture with monocytes and Borrelia in the absence or presence of either DMSO or z-VAD-fmk (50 µM).

 
These findings suggested that Borrelia stimulation of monocytes would generate caspase activity. The presence of caspase activity in Borrelia-activated monocytes was therefore more closely defined, initially using the tetrapeptide caspase substrate DEVD linked to rhodamine. Cleavage at the C-terminal aspartate residue releases the rhodamine to become fluorescently activated (41). Using this assay, it was apparent that freshly isolated monocytes had no detectable caspase activity, whereas monocytes stimulated 48 h earlier with B. burgdorferi manifested readily detectable caspase activity (Fig. 7A). The levels of caspase activity were even more pronounced in myeloid DC that were derived from monocytes following culture in GM-CSF and IL-4. The findings using DEVD-rhodamine were further confirmed through direct labeling of only active caspases using biotin-labeled z-VAD-fmk. This allowed the precipitation with avidin-Sepharose of selectively the active caspase fraction, as we have previously shown in activated T cells (41). This assay revealed that freshly isolated monocytes did not have detectable active caspase-8 (Fig. 7B). However, DC manifested easily detectable caspase-8 (Fig. 7B). Thus, the transition from resting monocyte to immature myeloid DC in the presence of GM-CSF and IL-4 results in the induction of caspase activity, which is critical for certain effector functions of these cells.


Figure 7
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  		FIGURE 7. Caspase activity in Borrelia-activated monocytes and DC. A, Caspase activity was measured using the DEVD-rhodamine assay in CD14+ monocytes either freshly isolated or after 48 h stimulation with B. burgdorferi, and compared with myeloid DC derived from monocytes using GM-CSF plus IL-4. B, Active caspase-8 in DC. Cells were lysed in buffer containing biotin-VAD-fmk and active caspases selectively precipitated using avidin-Sepharose and then immunoblotted for caspase-8. Whole cell lysates (WCL) from fresh monocytes was included as a positive control for caspase-8 staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The current results support a model in which synovial {gamma}{delta} T cells of the V{delta}1 subset are activated indirectly by B. burgdorferi via TLR signals, in part by TLR2. The findings are consistent with earlier reports that Borrelia lipopeptides bind TLR2 and this required the tripalmitic acid side chains (37). We also observed that removal of the fatty acid moieties eliminated the ability of OspA to induce CD25 expression on the V{delta}1 cells. This may extend to other TLR ligands and demonstrate a novel link between the innate and adaptive immune responses. The fact that the findings also apply to the activation of murine {gamma}{delta} T cells serves to enhance the validity of these results. We found no evidence for surface expression of TLR2 by the V{delta}1 cells themselves, nor evidence for direct activation of the {gamma}{delta} T cells by B. burgdorferi, whereas TLR2 is abundantly expressed by monocytes and DC. Thus, the current evidence supports a model in which activation of V{delta}1 cells by B. burgdorferi is not Ag-specific, but rather, indirect, in which Borrelia stimulates APCs to up-regulate a ligand(s) for the V{delta}1 TCR. The ligand for the TCR-V{delta}1 is currently unknown and is the subject of active investigation.

The predominant human {gamma}{delta} T cell subset in peripheral blood is V{gamma}2V{delta}2, which reacts to nonpeptide Ags from Mycobacterium. These include isoprenyl pyrophosphates (27, 28, 29, 30), as well as alkylamine Ags (31). These are products of microbes as well as self-Ags. By contrast, the V{delta}1 subset is resident in the intestine (21) and accumulates at sites of inflammation, such as the synovium in rheumatoid arthritis (19) and Lyme arthritis (20). Little is known regarding the specificity of V{delta}1 cells. A report has suggested that some V{delta}1 cells react to CD1c (38), and that murine V{gamma}4+ T cells respond to CD1d during coxsackievirus infection (39). We have not observed any suggestion of CD1c reactivity by our Lyme arthritis synovial V{delta}1 cells (C. Collins, unpublished observations). There is, nonetheless, a suggestion that the synovial TCR-V{delta}1 determinant may indeed be nonpolymorphic, given that the V{delta}1 clones can be activated by B. burgdorferi using monocytes or DC from HLA mismatched donors. Most human {gamma}{delta} T cells, including the synovial V{delta}1 cells, express the activating NK receptor, NKG2D, which binds MHC class I chain-related gene A, a nonpolymorphic MHC class I-related molecule (42, 43). However, the DC used in these studies expressed no detectable surface MICA. Nonetheless, the transwell studies indicate that cell contact is required with DC to activate the V{delta}1 cells.

It is also possible that DC- and monocyte-derived cytokines may potentiate activation of {gamma}{delta} T cells, either synergistically with, or independently of TCR signals. Similar indirect cytokine signaling has been observed in other T cell subsets such as NKT cells (44) and during homeostatic proliferation of T cells (45). In addition, the main cytokines induced by Borrelia stimulation of monocytes or DC (IL-1β, IL-6, and TNF-{alpha}) have been observed to augment proliferation of CD4+ {alpha}β T cells (46). However, we could not detect any significantly augmented CD25 expression by the addition of these cytokines alone or together, nor any reduced CD25 induction by blocking Abs to these cytokines. We have also previously observed that the activation of the V{delta}1 clones by DC and Borrelia is blocked by anti-TCR-{gamma}{delta} but not by blocking Abs to MHC class I or class II, indicating that the TCR-{gamma}{delta} is involved with this response, but not MHC classical class I or class II molecules (35). The collective findings thus suggest that DC activate {gamma}{delta} T cells primarily via one or more surface determinants.

In addition to its proapoptotic function in death receptor pathways, caspase-8 has been noted to be critical for proliferation of T cells and a growing number of other cell types (47, 48, 49). Humans bearing a germline point mutation of caspase-8 manifest a defect in T, B, and NK cells (50). More recently it was discovered that caspase-8 is also important for certain types of TLR signaling. B cells lacking caspase-8 were observed to have an attenuated proliferation and Ab response following signaling of TLR3 or TLR4 (40). We now observe that Borrelia stimulates caspase activity in fresh human monocytes, and this is required to produce certain cytokines, up-regulate surface CD1 family members, as well as being able to activate V{delta}1 T cells. The active caspase complex in DC contains caspase-8. These findings thus begin to form a signaling pathway between certain TLR and an active caspase complex that may contain components common to other receptor signaling that leads to NF-{kappa}B activation.

We recently reported that synovial {gamma}{delta} T cells were capable of activating DC through a Fas/FasL mechanism (33). In this system, V{delta}1 cells were observed to express high and sustained levels of FasL, whereas myeloid DC were highly resistant to Fas-induced cell death due to their high expression of the caspase-8 inhibitor, c-FLIPL (33). Furthermore, the augmented expression of c-FLIPL was able to divert Fas signals from caspase-8 activation and toward activation of NF-{kappa}B and stimulation by DC of cytokine production and expression of surface costimulatory molecules (33). Combined with the current findings, a model emerges in which synovial V{delta}1 T cells and myeloid DC can mutually stimulate each other in the presence of B. burgdorferi through a process initiated by Borrelia engagement of TLR signaling. The products of this interaction are also important for activation of the adaptive immune response, placing certain {gamma}{delta} T cells at a juncture between the innate and adaptive immune responses.


   Acknowledgments
 
We thank Ms. Colette Charland for technical assistance with flow cytometry, Dr. Matthew Poynter of the Vermont Lung Center for the kind gift of TLR2–/–, TLR9–/–, and MyD88–/– mice, and Dr. Robert Finberg for the blocking anti-TLR2 Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AR43520 and AI 45666 (to R.C.B.), and P30CA22435 (Vermont Cancer Center) from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Ralph C. Budd, Immunobiology Program, The University of Vermont College of Medicine, Given Medical Building, D-305, Burlington, VT 05405-0068. E-mail address: ralph.budd{at}uvm.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived dendritic cell. Back

Received for publication March 6, 2008. Accepted for publication June 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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