Expansion of human {gamma}{delta} T cells after in vitro stimulation with Campylobacter jejuni

Ildiko Van Rhijn1, Leonard H. Van den Berg1, C. Wim Ang3, Joke Admiraal4 and T. Logtenberg2

Departments of 1 Neurology and 2 Immunology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands 3 Departments of Neurology and Immunology, Erasmus University Rotterdam, 3000 VB, Rotterdam, The Netherlands 4 Rijksinstituut voor Volksgezondheid en Milieu, 3720 BA, Bilthoven, The Netherlands

Correspondence to: L. H. Van den Berg; E-mail: l.h.vandenberg{at}neuro.azu.nl
Transmitting editor: K. Sugamura


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Campylobacter jejuni is currently the prime cause of food-borne bacterial gastro-enteritis. An important complication of C. jejuni enteritis is Guillain–Barré syndrome (GBS), an immune-mediated disorder of peripheral nerve tissue. Because little is known about T cell reactivity to C. jejuni, we have analyzed the in vitro immune response of normal individuals against five isolates of C. jejuni representing five different serotypes. We found a preferential expansion of peripheral blood {gamma}{delta} T cells after exposure to crude sonicates of all five C. jejuni serotypes. Expansion of {gamma}{delta} T cells was dependent on the presence of CD4+/{alpha}ß+ T cells in the cultures or addition of exogenous IL-2 or IL-15. C. jejuni stimulation was mediated via the TCR and appeared to be induced by a non-proteinaceous bacterial antigen, most likely of phosphoantigenic origin.

Keywords: bacterial serotype, CD4+ {alpha}ß T cells, Guillain–Barré syndrome, IL-2, IL-15


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Campylobacter jejuni is the leading cause of bacterial food-borne diarrheal disease throughout the world (1). Elevated serum IgG antibodies against C. jejuni, suggestive of previous exposure to C. jejuni, can be found in 90% of the normal population (2). Humans are usually infected by consumption of contaminated poultry, milk products or water, frequently resulting in inflammatory gastro-enteritis in naive individuals. An important complication of C. jejuni enteritis is Guillain–Barré syndrome (GBS), an acute inflammatory demyelinating polyneuropathy which is the leading cause of acute flaccid paralysis in western countries. It has been postulated that the pathogenesis of GBS involves molecular mimicry because sera of patients with C. jejuni-associated GBS have high titers of antibodies against peripheral nerve gangliosides (35) and the lipopolysaccharide (LPS) from various C. jejuni serotypes contains a terminal tetrasaccharide identical to that of the GM1 or GQ1b ganglioside (6). It has been suggested that these cross-reactive antibodies mediate the immune-related destruction of peripheral nerve tissue in GBS and that the diversity of clinical features may relate to the serotype of C. jejuni (7). Serotypes O:19 (8,9) and O:41 (7) are over-represented in GBS patients in Japan and South Africa respectively.

Although the humoral immune response to C. jejuni and the occurrence of cross-reactive antibodies to peripheral nerve tissues has been addressed in many studies, relatively little is known about the role of T lymphocytes in GBS. Histological studies of peripheral nerve from patients with GBS show evidence for T lymphocytes infiltrating the affected tissue (1012), and evidence for the presence of activated T cells and T cell products in the blood and serum has been found (1315). Interestingly, a T cell line established from a nerve biopsy of a patient with GBS with preceding C. jejuni infection was shown to consist entirely of T cells expressing the {gamma}{delta} TCR (16). In vitro stimulation of blood mononuclear cells from GBS patients, gastroenteritis patients and normal volunteers with C. jejuni yielded T cell lines that were enriched for {gamma}{delta} T cells (17).

As a prelude to studies on the role of C. jejuni-reactive T cells in patients with GBS, we have analyzed the in vitro cellular immune response of normal individuals against five isolates of C. jejuni representing five serotypes, and obtained from both GBS patients and uncomplicated enteritis patients.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bacterial strains and antigen preparation
A C. jejuni strain was isolated from the stool of a GBS patient and was serotyped as O:2 according to the system described by Penner (18). C. jejuni strains with serotypes O:1 (43429), O:18 (43446) and O:19 (43445) were obtained from ATCC (Manassas, VA). A strain with serotype O:4 (10938) was from the Culture Collection University of Göteborg (Göteborg, Sweden). C. jejuni strains were cultured under micro aerobic conditions, at 37°C, on Blood Free Campylobacter Selective Agar Base (Oxoid, Basingstoke, UK) supplemented with Cefoperazone (32 mg/l) and Amphotericine B (5 mg/l). Bacteria were washed twice in PBS and heated at 100°C for 40 min, followed by probe sonication for 2 min. The protein concentration of the preparation was assessed by the DC protein assay (Bio-Rad, Hercules, CA).

LPS was extracted from C. jejuni by the phenol–water method as described by Westphal (19). In short, crude antigen preparations were extracted at 100°C in a mixture of water and phenol. The aqueous phase was dialyzed against water, centrifuged at 300,000 g for 2 h and freeze-dried. The pellet was weighed and dissolved in water at a concentration of 1 mg/ml. The LPS was analyzed by SDS–PAGE as described below. The protein concentration of the preparation was assessed by the DC protein assay (Bio-Rad). Prepar ations that contained no detectable protein were used for T cell stimulation studies.

Aliquots of delipidated preparations of C. jejuni containing 150 µg protein were treated twice with 1 µg proteinase K (Boehringer Mannheim, Almere, The Netherlands) for 30 min at 37°C and for 5 min at 70°C. Subsequently, 1 µg pronase E (Boehringer-Ingelheim, Heidelberg, Germany) was added and incubated at room temperature for 1 h. The enzymes were finally inactivated at 100°C for 5 min. The effectivity of the protease treatment was checked by SDS–PAGE.

Isopentenyl pyrophosphate (IPP; Sigma, St Louis, MO) was dried under a stream of nitrogen, dissolved in medium by water bath sonication and used at 15 µg/ml.

SDS–PAGE
SDS–PAGE was performed on 14% polyacrylamide gels using the Mini Protean II system of Bio-Rad. One-hour electroblotting on nitrocellulose membrane (Bio-Rad) was carried out using the Mini Transblot system (Bio-Rad). Antigen preparation containing 15 µg protein, the equivalent amount of protease-digested antigen preparation, or 1 µg LPS was run per lane. Blots were stained with Coomassie Brilliant blue for protein detection or with a silver stain kit (Novex, San Diego, CA), modified after Tsai (20) for detection of LPS.

Determination of anti-C. jejuni antibodies in serum
Serum from normal donors was used in serial dilutions in an ELISA to determine the presence of IgM, IgG and IgA anti-C. jejuni antibodies (2). Ratios were obtained by comparing with negative reference serum. IgM or IgA ratios >1 were considered to be indicative of recent C. jejuni infection and IgG ratios of >2 in the absence of IgM or IgA antibodies indicative of previous C. jejuni infections in a more distant past (2).

T cell cultures and proliferation assays
For proliferation assays, peripheral blood mononuclear cells (PBMC) from normal donors lacking IgM or IgA anti-C. jejuni antibodies but with IgG ratios >2 were used. PBMC were isolated from heparinized blood by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density centrifugation, and resuspended in culture medium consisting of RPMI 1640 (Life Technologies, Paisley, UK) supplemented with L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin and 5% heat-inactivated pooled human AB serum. For proliferation assays, serial 7-fold dilutions of antigen were added to triplicate wells in round-bottom 96-well microtiter plates containing 105 cells. At day 5 (PBMC) or day 3 (clones) of culture, 1 µCi [3H]thymidine was added. After 18 h cells were harvested onto filters and counted in a ß-counter. PBMC were cultured for 12 days prior to flow cytometric analysis. The final antigen concentration in these T cell cultures corresponded to 3 µg protein/ml. For inhibition assays, 20 µg/ml mAb against the {gamma}{delta} TCR, IL-2 or IL-15 was added to the cultures at days 1, 3 and 6. Human IL-2 was obtained from Strathmann Biotech (Hannover, Germany) and used at 10 U/ml, and IL-15 was from PeproTech (Rocky Hill, NJ) and used at 10 ng/ml.

Cloning and re-stimulation was performed at 17 days after the last stimulation, using irradiated allogeneic PBMC as feeder cells, 10 ng/ml IL-15 and antigen at 3 µg protein/ml. Note that phytohemagglutinin was not used. Proliferation assays were performed without cytokines.

Cell depletion and sorting
For depletion experiments, PBMC from individuals with {gamma}{delta} T cells that were >90% CD4/CD8 were used. CD4+ or CD8+ cells were removed from PBMC by incubation with anti-CD4 or anti-CD8 antibody and magnetic M450 Dynabeads coated with goat anti-mouse IgG (Dynal, Oslo, Norway), according to the manufacturer’s protocol. CD4+, CD14+, CD16+, CD19+, CD33+, V{alpha}24 and {alpha}ß TCR+ populations were depleted from PBMC by cell sorting on a FACStar Plus cell sorter (Becton Dickinson, San Jose, CA). PBMC populations depleted of a population of cells by magnetic beads or cell sorting contained <1% of the depleted population as determined by flow cytometry.

mAb and flow cytometry
Anti-human IL-2 (clone B-G5) and IL-15 (clone 34593.11) mAb for use in cell culture were purchased from Diaclone (Besancon, France) and R & D systems (Minneapolis, MN) respectively. The anti-{gamma}{delta} TCR mAb producing hybridoma 5a6.e9 was obtained from the ATCC. Hybridoma culture supernatant was purified on a Protein A column (Bio-Rad), according to the manufacturer’s protocol. Isotype control Ig was obtained from ICN (Eschwege, Germany). For flow cytometry, anti-CD3 mAb conjugated to phycoerythrin (PE)–Cy5 and unconjugated anti-V{alpha}24 were obtained from Immunotech (Marseille, France), unconjugated anti-V{delta}1 (clone A13) was a gift of Dr L. Moretta, (Genova), unconjugated anti-V{delta}2 (clone 4G6), anti-V{gamma}4 (clone 4A11) and anti-V{gamma}9 (clone B3) were gifts of Dr G. De Libero (Basel), unconjugated anti-CD4 was from Dako (Glostrup, Denmark), goat anti-mouse–PE was from Southern Biotechnology (Birmingham, AL), and all other antibodies were from Becton Dickinson (San Jose, CA). Freshly isolated or cultured cells (105) from 24 pooled wells were used for each staining. Washing of cells and dilutions of antibodies were carried out with PBS containing 1% BSA. The unconjugated antibodies were used undiluted and removed by washing before incubation with goat anti-mouse–FITC (1:50) or goat anti-mouse–PE (1:500). Cells were washed again and incubated with normal mouse serum (1:1000) before anti-CD3–PE–Cy5 was added. Flow cytometric analysis was performed on a FACScan (Becton Dickinson).

Calculation of the T cell expansion index
The expansion of {gamma}{delta} T cells was monitored by flow cytometric analysis as described (21) with minor modifications. Stimulated cells were double-stained with anti-{gamma}{delta} TCR or anti-{alpha}ß TCR antibodies in combination with an anti-CD3 antibody, and the frequency of {gamma}{delta} or {alpha}ß cells in the CD3 and life gate was determined. The absolute number of {gamma}{delta} or {alpha}ß T cells was calculated by multiplying the total number of viable cells in a culture by the frequency of {gamma}{delta} or {alpha}ß T cells. The expansion index was calculated by dividing the absolute number of {gamma}{delta} or {alpha}ß T cells at day 12 of culture by the absolute number of {gamma}{delta} or {alpha}ß T cells seeded at the onset of culture.

PCR and nucleotide sequence analysis of TCR V{delta} regions
Cultures with expanded populations of TCR V{delta}2 T cells after stimulation with the C. jejuni O:1 antigen were harvested, processed for nucleotide sequence analysis of expressed V{delta}2 genes and compared to nucleotide sequences expressed in TCR V{delta}2 T cells at the onset of the culture. RNA extraction was performed with Dynabeads Oligo(dT)25 (Dynal, Oslo, Norway) according to the manufacturer’s protocol, followed by two washes in 1 x AMV-RT buffer (Boehringer Mannheim). After the last wash 1 µl RNAguard, 8.5 µl water, 5 µl DTT (100 mM), 5 µl dNTP (10 mM), 5 µl 5 x AMV-RT buffer and 0.5 µl AMV-RT were added, and incubated at 42°C for 90 min for cDNA synthesis.

PCR conditions and primers were identical to those described previously (22,23). PCR products were ligated in the pGEM-T Vector (Promega, Madison, WI) and transformed into Escherichia coli. Plasmid DNA was isolated from randomly picked bacterial colonies and used for nucleotide sequence analysis. The T7 promoter primer was used for sequencing with the Terminator Ready Reaction DyeDeoxy Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems, Foster City, CA). Samples were analyzed by automated fluorescence detection.

Clones that were not recognized by the anti-V{delta}2 antibody 4G6, nor by the anti-V{delta}1 antibody A13, were analyzed by V{delta}2 PCR as described. Amounts of cDNA were standardized in a ß-actin PCR.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Crude preparations of C. jejuni induce vigorous T cell proliferation
PBMC from four different normal donors, with no serological evidence of a recent C. jejuni infection, were stimulated in vitro with serial dilutions of crude antigen preparations of C. jejuni isolates of five different serotypes. Representative dose–response curves of a single donor stimulated with the five C. jejuni serotypes and of four donors responding to a single C. jejuni serotype are shown in Fig. 1. In all experiments, significant proliferation (>3 times the background of non-stimulated cells) was observed at protein concentrations as low as 8.7 ng/ml or less. Maximum proliferation was observed at 3 or 21 µg/ml. Using [3H]thymidine incorporation as a read-out, only minor differences in the dose–response curves upon stimulation with different C. jejuni serotypes were observed (Fig. 1). In 40 individuals analyzed to date, we have found only a single individual that completely and repeatedly failed to respond to all five C. jejuni isolates (results not shown).



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Fig. 1. (A) [3H]Thymidine incorporation after stimulation of PBMC from donor M with different concentrations of crude C. jejuni preparations of five different Penner serotypes. (B) Variability in the responses of donors T, Z, M and I to stimulation with the O:19 strain.

 
Crude preparations of C. jejuni induce selective outgrowth of {gamma}{delta} T cells
For identification of the proliferating cells in the cultures stimulated with C. jejuni crude antigen preparations, PBMC at the onset and after 12 days of culture were double-stained with {gamma}{delta} or {alpha}ß TCR-specific antibodies in combination with an anti-CD3 mAb and analyzed by flow cytometry. The expansion index was calculated by dividing the absolute numbers of {gamma}{delta} T cells before and after stimulation, and provides a direct measure of T cell expansion during culture. In all donors tested, the frequency of circulating {gamma}{delta} T cells was in the normal range, varying between 0.5 and 10% of the total CD3+ T cell population (not shown). Selective outgrowth of {gamma}{delta} cells and minimal or no outgrowth of {gamma}{delta} T cells was induced upon stimulation of PBMC of four normal donors with crude preparations of all five isolates of C. jejuni. A representative flow cytometric analysis of T cells before and after cell culture is shown in Fig. 2(A). The results of these experiments, expressed as {gamma}{delta} or {alpha}ß T cell-specific expansion indices, are summarized in Table 1. The {gamma}{delta} T cell-specific expansion index ranged from 2 to 151 (median 11.5), whereas the expansion index of {alpha}ß T cells in the cultures was significantly lower, ranging from 0.8 to 2.9 (median 1.6).



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Fig. 2. Flow cytometric analysis of PBMC of donors Z and M at the onset and after 12 days of culture with C. jejuni. Cells from triplicate or more wells were pooled and double-stained with the CD3–PE–Cy5 and pan-{gamma}{delta} TCR-F mAb (A), or with the CD3–PE–Cy5 in combination with the V{delta}1-F, V{delta}2-F, V{gamma}4-F or V{gamma}9-F antibodies (B). Percentages of {gamma}{delta} T cells are expressed as percentages within the live, CD3+ population. Note that the sum of the two V{gamma} families does not match the total amount of {gamma}{delta} T cells of donor M at day 12, as determined by the pan {gamma}{delta} TCR antibody.

 

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Table 1. Expansion indices of {alpha}ß and {gamma}{delta} T cells after 12-day culture of PBMC of four normal donors with crude preparations of five different C. jejuni isolates
 
V{gamma} and V{delta} chain utilization in {gamma}{delta} T cells stimulated with C. jejuni
TCR V{gamma} and V{delta} utilization among {gamma}{delta} T cells before and after stimulation with crude preparations of C. jejuni was analyzed by flow cytometry using mAb specific for V{gamma}4, V{gamma}9, V{delta}1 and V{delta}2, and a pan-{gamma}{delta} TCR mAb. In three of the four donors analyzed (T, R and Z), the number of (V{delta}1+ plus V{delta}2+) and (V{gamma}4+ plus V{gamma}9+) cells matched the number of {gamma}{delta} T cells detected with the pan-{gamma}{delta} TCR antibody. The results obtained with donor Z are shown in Fig. 2(upper panels). In donor M, the number of {gamma}{delta} T cells detected with the pan-{gamma}{delta} TCR antibody exceeded the sum of the individual V{gamma} and V{delta} families, suggesting the expansion of {gamma}{delta} T-expressing V regions not detected with the available mAb (Fig. 2, lower panels).

In donors T, Z and I, prior to culture 95, 56 and 53% of the peripheral blood {gamma}{delta} T cells respectively expressed a TCR encoded by the V{delta}2 and V{gamma}9 gene segments. After culture with all five serotypes of C. jejuni, the V{delta}2/V{gamma}9 combination was expressed by virtually all {gamma}{delta} T cells. A representative experiment is shown in Fig. 3. In donor M, a different pattern was observed. In this donor we observed outgrowth of T cells expressing V{delta}1 in combination with a V{gamma} gene segment not identified with our mAb after stimulation with four of the serotypes and outgrowth of an undetermined V{delta} in combination with V{gamma}9 after stimulation with the fifth serotype. {gamma}{delta} T cells in the non-stimulated PBMC of this donor expressed TCR encoded by V{delta}2 (24%) and V{delta}1 (76%) showing that T cells expressing the undetermined V{delta} comprised only a minor population at the onset of culture. PCR analysis of RNA extracted from {gamma}{delta} T cells clones that were not recognized by the anti-V{delta}1 and the anti-V{delta}2 antibodies showed that they all express a V{delta}2 chain (not shown).



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Fig. 3. V{gamma} and V{delta} gene utilization in {gamma}{delta} T cells before and after in vitro stimulation with C. jejuni. V gene families showing <5% increase after the cultures are not shown. Representative data of the PBMC of two donors stimulated with three different crude C. jejuni serotypes are shown.

 
Expanded {gamma}{delta} T cells of all donors stimulated with all bacterial strains were mostly CD4/CD8 with a variable percentage (range 0–35%) of CD4/CD8+ {gamma}{delta} T cells (not shown).

Nucleotide sequence analysis of TCR V{delta} regions
We assessed the clonal diversity of {gamma}{delta} T cells proliferating in response to in vitro stimulation with C. jejuni. Freshly isolated PBMC from donors R and Z were compared to cultures that had been stimulated for 12 days with the crude antigen preparation of C. jejuni strain O:1. The cells were used for RNA extraction and first-strand cDNA synthesis, and amplified with a V{delta}2-specific primer pair. PCR fragments were cloned and the nucleotide sequence of 80 independent V{delta}2 inserts was obtained comprising 40 inserts from each donor, equally divided between PBMC before and after culture. The V{delta}–D{delta}–J{delta} junctional sequences are shown in Table 2. All V{delta}2 sequences analyzed were in-frame. In the collections of 20 sequences from freshly isolated PBMC, all sequences from donor Z and 19/20 sequences from donor R were unique. After culture, the collections of V{delta}2 sequences of the two donors (R and Z) showed clear evidence of oligoclonal expansion as evidenced by the recurrence of identical V{delta}–D{delta}–J{delta} junctional sequences in independently picked bacterial clones.


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Table 2.
 
The C. jejuni antigen with strong {gamma}{delta} T cell-stimulatory capacity is a protease-resistant compound
Human {gamma}{delta} T cells have been reported to respond to a wide variety of antigens. To determine the nature of the antigen with strong {gamma}{delta} T cell-stimulatory capacity, crude C. jejuni preparations were subjected to a number of treatments and separation procedures.

LPS, known to stimulate murine {gamma}{delta} T cells (24), was isolated from C. jejuni isolates O:1 and O:2 by phenol–water extraction and identified on silver-stained SDS–PAGE gels (Fig. 4, right panel). In dose–response curves with concentrations ranging from 1.2 ng/ml to 3 µg/ml, LPS induced a just-above background proliferative response in 12-day cultures of PBMC. At day 12 of culture, no {gamma}{delta} T cells were detectable by flow cytometry (results not shown), indicating that LPS is not the compound responsible for the {gamma}{delta} T cell response to C. jejuni antigen preparation.



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Fig. 4. Protease-treated (left panel, left lane) and untreated (left panel, right lane) C. jejuni O:19 antigen preparation on a Coomassie stained blot, and phenol–water extracted LPS of O:1 and O:19 on a silver stained gel (right panel).

 
The crude antigen preparations of serotypes O:8 and O:19 were delipidated, treated with pronase E and proteinase K, and analyzed by SDS–PAGE to monitor the efficiency of protein degradation. After blotting and Coomassie Brilliant blue staining, no protein could be detected (Fig. 4, left panel, left lane). PBMC from a normal donor were stimulated with mock-treated or protease-treated antigen preparations, and a [3H]thymidine incorporation assay and flow cytometric analysis were performed. No differences in the proliferation-inducing capacity between the mock-treated and the delipidated/protease-treated C. jejuni preparations were observed (Fig. 5A). Flow cytometric analysis showed that stimulation of PBMC with either preparation resulted in the selective and extensive expansion of {gamma}{delta} T cells (Fig. 5B).



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Fig. 5. [3H]Thymidine incorporation (A) and FACS analysis (B) of non-stimulated PBMC of donor R (left panel) and PBMC stimulated with mock-treated (middle panel) or protease-treated/delipidated serotype O:19 C. jejuni preparations (right panel). Both mock and protease-treated/delipidated preparations induced selective outgrowth of {gamma}{delta} T cells, as shown by flow cytometric analysis of CD3–PE–Cy5/pan-{gamma}{delta} TCR-F stained cells (b).

 
Reactivity of {gamma}{delta} T cell clones and C. jejuni-stimulated cell lines
We generated polyclonal cell lines from PBMC of three different donors by two cycles of stimulation with C. jejuni antigen. Clones were obtained from these cell lines, and polyclonal lines and clones were tested for reactivity with C. jejuni or IPP. All cell lines proliferated in response to stimulation with C. jejuni and IPP. These results were confirmed by the proliferative response of three independent clones from two of the donors (Fig. 6).



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Fig. 6. Proliferation of a polyclonal cell line and three independent clones to C. jejuni and IPP stimulation. Clones m2 and d62 are derived from different donor, and are V{delta}2+ as they stain with the 4G6 antibody, and clone m31 gives a positive V{delta}2 PCR signal although it does not stain with the available antibodies. The stimulation index is defined as the c.p.m. obtained from wells with responder cells, irradiated feeder cells and antigen, divided by the c.p.m. from the wells with responder cell and irradiated feeder cells without antigen.

 
The presence of CD4+/{alpha}ß T cells is a prerequisite for the {gamma}{delta} T cell response to C. jejuni
To determine whether signaling via the {gamma}{delta} T cell TCR was essential for the observed stimulation of {gamma}{delta} T cells by C. jejuni, we administered a blocking anti-{gamma}{delta} TCR antibody (25) to the cultures. As shown in Fig. 7, control antibody does not influence the response of {gamma}{delta} T cells to C. jejuni, whereas the anti-{gamma}{delta} TCR 5a6.e9 mAb completely abolishes {gamma}{delta} T cell expansion.



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Fig. 7. The {gamma}{delta} T cell response to stimulation with C. jejuni serotype O:18 is inhibited by addition of 20 µg/ml anti-{gamma}{delta} TCR mAb, but not by isotype-matched control mAb. The {gamma}{delta} T cell expansion index of different donors in response to C. jejuni O:18 stimulation is depicted.

 
It has been shown that {gamma}{delta} T cell expansion of PBMC upon stimulation with Daudi cells (26) or Mycobacterium tuberculosis (27,28) depends on the presence of CD4+ cells. To determine the contribution of CD4+ cells to the response to C. jejuni, PBMC were depleted with CD4 or CD8 mAb-coated magnetic beads and subsequently stimulated with C. jejuni. Depletion of CD8+ cells did not affect the proliferative response to C. jejuni, whereas depletion of CD4+ cells completely abrogated {gamma}{delta} T cell expansion (Fig. 8). These findings were extended by depleting PBMC of a number of cell subpopulations by cell sorting. Thus, depleting PBMC of TCR{alpha}ß+ T cells or CD4+ cells completely abrogated the proliferative response, whereas depletion of CD14+/CD16+, CD33+, CD19+ or V{alpha}24+ cells had no effect (Fig. 9).



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Fig. 8. Immunomagnetic depletion of CD4+ cells, but not of CD8+ cells, from PBMC of two different donors abrogated the response to C. jejuni O:19 antigen (a) and O:2 antigen (b). The response could be restored by the addition of 10 U/ml IL-2 (a and b) or 10 ng/ml IL-15 (b). Dashed bars represent the {gamma}{delta} T cell-specific expansion index and open bars represent the {alpha}ß T cell-specific expansion index. The results are representative of experiments with PBMC from eight different donors.

 


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Fig. 9. The {gamma}{delta} T cell expansion indices of C. jejuni strain O:1-stimulated PBMC from donor A and B, depleted for {alpha}ß TCR+, CD4+, CD14+/CD16+, CD33+ and CD19+ cells by cell sorting. As a control for the sorting procedure, unstained PBMC were run through the sorter and put in culture. In separate experiments with PBMC from donors C and D, the NKT cell population was depleted from the PBMC with an anti-V{alpha}24 antibody (right panels).

 
The {gamma}{delta} T cell response to C. jejuni can be restored by adding IL-2 or IL-15 to CD4-depleted cultures of PBMC
The role of the T cell growth factors IL-2 and IL-15 was assessed by adding these cytokines to CD4-depleted cultures of PBMC stimulated with crude C. jejuni preparations. Low doses of IL-2 or IL-15 were capable of restoring the {gamma}{delta} T cell response in CD4-depleted populations (Fig. 8). As noted previously by others (29,30), {gamma}{delta} T cells from some donors proliferated in response to stimulation with IL-2 alone, in the absence of C. jejuni antigen (results not shown). We did not include these donors in these experiments.

mAb to IL-2 and IL-15 unveil a dichotomy in cytokine production in cultures of C. jejuni-stimulated PBMC
The observation that low doses of IL-2 and IL-15 restored the {gamma}{delta} T cell response to C. jejuni in CD4-depleted PBMC populations was further supported by the finding that anti-IL-2 mAb, and, in some donors, the combination of anti-IL-2 and anti-IL-15 antibodies blocked the {gamma}{delta} T cell response to C. jejuni when total PBMC were stimulated (Fig. 10). Anti-IL-15 antibodies alone did not affect the {gamma}{delta} T cell response in six donors tested.



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Fig. 10. Inhibition of the ex vivo {gamma}{delta} T cell response by anti-IL-2 mAb (donor L) or a combination of anti-IL-2 and anti-IL-15 mAb (donor S). The {gamma}{delta} T cell expansion index of different donors in response to C. jejuni O:18 stimulation is depicted. Results are representative of experiments with PBMC from six donors.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
C. jejuni has become recognized as the most frequent antecedent pathogen associated with the development of GBS. Although the humoral immune response to C. jejuni and the occurrence of cross-reactive antibodies to peripheral nerve tissues has been addressed in many studies, relatively little is known about the role of T lymphocytes in GBS. We therefore analyzed the human T cell response to crude preparations of five different serotypes of C. jejuni.

The results presented here show that all five different serotypes of C. jejuni induce in vitro proliferation of {gamma}{delta} T cells present in PBMC from normal individuals. In 40 individuals analyzed to date, we found only a single individual that completely and repeatedly failed to respond to all five C. jejuni serotypes.

In three out of four donors extensively analyzed here, stimulation with all five C. jejuni serotypes resulted in the expansion of T cells expressing the V{gamma}9/V{delta}2 TCR. In the fourth donor, proliferation of T cells expressing V{delta}1 and an undetermined V{gamma} was observed. The V{gamma}9/V{delta}2 TCR pair is utilized in 50–75% of circulating {gamma}{delta} T cells, whereas the second major population of blood {gamma}{delta} T cells expresses V{delta}1 in combination with one of several V{gamma} gene segments (31). Expansion of circulating V{delta}1-expressing T cells has only occasionally been reported in the context of HIV (32,33) and cytomegalovirus after renal transplant (34). V{gamma}9/V{delta}2 T cell expansions are associated with many human infectious diseases including Epstein–Barr mononucleosis (35), cytomegalovirus disease (36), tuberculosis (37), tularemia (38), listeriosis (39), toxoplasmosis (40), salmonellosis (41) and infection with Coxiella burnetii (42). Epstein–Barr and cytomegalovirus are two pathogens that are also frequently found in association with GBS. It is possible that C. jejuni contains more than one class of {gamma}{delta} T cell antigen, capable of stimulating both classes of {gamma}{delta} T cells. Previous exposure to the same or cross-reacting antigens of a certain donor would influence the {gamma}{delta} T cell family growing out after in vitro stimulation with C. jejuni.

Previous reports have shown that the V{gamma}9/V{delta}2 T cells expanding in vitro in response to crude preparations of Mycobacterium tuberculosis show extensive V{delta}–D{delta}–J{delta} junctional diversity, reflecting a polyclonal T cell response (43,44). In contrast, nucleotide sequence analysis of 80 V{delta}2 regions of V{gamma}9/V{delta}2 T cells in the two donors analyzed here before and after stimulation with C. jejuni provided evidence for an oligoclonal T cell outgrowth.

Although {gamma}{delta} T cells do not need antigen-presenting cells like {alpha}ß T cells do, it has been previously shown that CD4+ T cells provide helper functions for the expansion of {gamma}{delta} T cells induced by mycobacterial extracts (27,28,45). Similarly, the proliferation of C. jejuni-reactive {gamma}{delta} T cells could be completely abrogated by depleting CD4+ T or {alpha}ß TCR+ T cells from PBMC. Depletion of other cell types including B cells, NK cells, dendritic cells, monocytes and NKT cells did not impair the ability of {gamma}{delta} T cells to respond to C. jejuni stimulation. The {gamma}{delta} T cell proliferation in CD4- or {alpha}ß TCR-depleted PBMC could be completely restored by addition of IL-2 to C. jejuni-stimulated cultures, confirming previous findings that, at least in vitro, IL-2 is a key cytokine for {gamma}{delta} T cell expansion provided by CD4+ T lymphocytes (27,46). Addition of IL-15, a T cell growth factor that shares some activities with IL-2 in vitro (47), also completely restored {gamma}{delta} T cell proliferation in CD4-depleted PBMC. This is in line with previous reports that show that IL-15 contributes to the activation {gamma}{delta} T cells (4850). Of note, recent data show that IL-15 may have a prominent role in stimulating and maintaining memory T cells in vivo in mice and humans (47,5153).

Blocking studies with mAb specific for IL-2 and IL-15 in cultures of unseparated PBMC stimulated with C. jejuni unveiled a dichotomy in cytokine production during culture of PBMC with crude lysates of C. jejuni. In three out of six donors tested, addition of anti-IL-2 mAb to the cultures completely inhibited {gamma}{delta} T cell proliferation, whereas addition of anti-IL-15 antibodies had no effect. In the cultures of PBMC of the remaining three donors analyzed, addition of anti-IL-2 or anti-IL-15 antibodies had no effect, whereas the simultaneous addition of both anti-IL-2 and anti-IL-15 antibodies completely inhibited the outgrowth of C. jejuni-reactive {gamma}{delta} T cells. Apparently, in all donors IL-2 is produced during culture of PBMC with C. jejuni, requiring at least the addition of anti-IL-2 antibodies to block {gamma}{delta} T cell proliferation. In cultures of PBMC of a subset of donors, IL-15 appears to be produced in addition to IL-2, requiring the presence of both anti-IL-2 and anti-IL-15 antibodies for inhibition. IL-15 may be produced by a variety of cell types including monocytes, dendritic cells and T cells [reviewed in (47)].

The depletion studies suggest that CD4+ T cells are the source of IL-2 and IL-15 in C. jejuni-stimulated cultures of PBMC. A possible mechanism of CD4+ T cell activation leading to cytokine production, but not proliferation, in the absence of antigen-presenting cells and protein antigens is activation via immune receptors recognizing more conserved molecules, like the Toll-like receptor family.

C. jejuni-specific stimulation was mediated via the {gamma}{delta} TCR. We demonstrated that protease treatment of crude preparations of C. jejuni did not diminish the {gamma}{delta} T cell response, further suggesting that conventional presentation of peptides by antigen-presenting cells was not required. Indeed, a number of non-proteinaceous compounds have been identified that stimulate {gamma}{delta} T cells (5459). Although the stimulatory component for the {gamma}{delta} T cells in the crude preparations of C. jejuni was not purified in the current studies, the fact that Campylobacter-reactive clones and cell lines recognize IPP suggests that the active compound in Campylobacter antigen preparation is of phosphoantigenic origin.


    Acknowledgements
 
This work was supported by ‘Het Prinses Beatrix Fonds’ (I. V. R.) and a fellowship of the Royal Netherlands Academy of Arts and Sciences (L. V. d. B.).


    Abbreviations
 
GBS—Guillain–Barré syndrome

IPP—isopentenyl pyrophosphate

LPS—lipopolysaccharide

PBMC—peripheral blood mononuclear cell

PE—phycoerythrin



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    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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