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The Journal of Immunology, 2007, 179, 6336 -6342
Copyright © 2007 by The American Association of Immunologists, Inc.
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Medline Plus Health Information
*Antibiotics
*Lyme Disease

Decline in the Frequencies of Borrelia burgdorferi OspA161–175-Specific T Cells after Antibiotic Therapy in HLA-DRB1*0401-Positive Patients with Antibiotic-Responsive or Antibiotic-Refractory Lyme Arthritis1

Priya Kannian*, Elise E. Drouin*, Lisa Glickstein*, William W. Kwok{dagger}, Gerald T. Nepom{dagger} and Allen C. Steere2,*

* Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; and {dagger} Benaroya Research Institute, Virginia Mason Medical Center, University of Washington, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Synovitis in patients with antibiotic-refractory Lyme arthritis persists for months to several years after antibiotic therapy. This course, which may result from infection-induced autoimmunity, is associated with T cell recognition of Borrelia burgdorferi outer surface protein A (OspA161–175) and with HLA-DR molecules that bind this epitope, including the DRB1*0401 molecule. In this study, we used tetramer reagents to determine the frequencies of OspA161–175-specific T cells in samples of PBMC and synovial fluid mononuclear cells (SFMC) from 13 DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory arthritis. Initially, three of the six patients (50%) with antibiotic-responsive arthritis and four of the seven patients (57%) with antibiotic-refractory arthritis had frequencies of OspA161–175-specific CD4+ T cells in peripheral blood above the cutoff value of 4 per 105 cells. Among the five patients with concomitant PBMC and SFMC, four (80%) had OspA tetramer-positive cells at both sites, but the mean frequency of such cells was 16 times higher in SFMC, reaching levels as high as 1,177 per 105 cells. In the two patients in each patient group in whom serial samples were available, the frequencies of OspA161–175-specific T cells declined to low or undetectable levels during or soon after antibiotic therapy, months before the resolution of synovitis in the two patients with antibiotic-refractory arthritis. Thus, the majority of patients with Lyme arthritis initially have increased frequencies of OspA161–175-specific T cells. However, the marked decline in the frequency of such cells with antibiotic therapy suggests that persistent synovitis in the refractory group is not perpetuated by these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lyme arthritis, which is caused by the tick-borne spirochete Borrelia burgdorferi (1), is characterized by intermittent or persistent arthritis in a few large joints, especially the knee, during a period of several years (2). This joint infection can usually be treated successfully with a 1-mo course of oral antibiotic therapy (3) or a 2- to 4-wk course of i.v. ceftriaxone (4), and the arthritis resolves (termed antibiotic-responsive arthritis). However, in rare cases patients have persistent arthritis despite treatment with ≥ 2 mo of oral antibiotics, ≥ 1 mo of i.v. antibiotics, or both (termed antibiotic-refractory Lyme arthritis) (3, 5). After such therapy, PCR results for B. burgdorferi DNA in joint fluid and synovial tissue are usually negative, suggesting that Lyme arthritis may persist after the near or total eradication of spirochetes from the joint with antibiotic therapy (6, 7).

Initial studies linked antibiotic-refractory Lyme arthritis with HLA-DR4 alleles (8) and with cellular and humoral immune responses to outer surface protein A (OspA)3 of B. burgdorferi (9, 10). The core immunodominant epitope of OspA presented by the DRB1*0401 molecule was identified as OspA165–173 (11), and patients with an antibiotic-refractory course often had T cell reactivity with this epitope (11, 12, 13, 14). Recently, antibiotic-refractory Lyme arthritis was associated with several HLA-DR molecules (primarily rheumatoid arthritis alleles) that bound the OspA165–173 epitope (15) including the DRB1*0401 molecule, which showed the strongest binding (15, 16). Thus, T cell reactivity with OspA165–173 may have a pathogenic role in antibiotic-refractory Lyme arthritis, but the mechanism is not yet clear.

To explain persistent synovitis after antibiotic treatment, four basic hypotheses have been proposed: 1) persistent infection; 2) retained spirochetal Ags; 3) infection-induced autoimmunity resulting from T cell epitope mimicry; or 4) infection-induced autoimmunity resulting from bystander activation of autoreactive T cells (17). If the numbers of T cells that recognize OspA remained high or increased in the postantibiotic period, it would imply that OspA Ag is still available and that these cells may be involved in the pathogenesis of antibiotic-refractory arthritis. Conversely, if the numbers of OspA-specific T cells declined in the postantibiotic period, it would suggest that OspA Ag is no longer available and that these cells are not perpetuating the arthritis. Thus, longitudinal analysis of the frequencies of OspA165–173-specific T cells in the infectious and postantibiotic periods might help to distinguish between these possibilities.

With the advent of class II MHC/peptide tetramers, direct quantitation and characterization of CD4+ T cells became possible (18). However, major hurdles remained because the frequencies and TCR binding avidity of Ag-specific CD4+ T cells may be low (19). In our initial study (20), we enumerated OspA164–175-specific CD4+ T cells in single samples obtained from joint fluid or peripheral blood in six DRB1*0401-positive patients with antibiotic-refractory arthritis using a DRB1*0401 class II tetramer covalently linked with the OspA peptide. With this methodology, two of three samples of synovial fluid mononuclear cells (SFMC) but none of six samples of PBMC had OspA164–175-specific T cell frequencies greater than those found with control tetramers. Thus, a more sensitive approach was needed.

In the present study we used MHC class II tetramer reagents to determine the frequencies of OspA161–175-specific T cells in a unique collection of single or serial samples of PBMC and SFMC from 13 DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis seen during the past 18 years. Using a newer staining protocol, the detection limit of OspA161–175-specific T cells was 4 per 105 CD4+ T cells, which made it possible to assess the frequency of these cells in PBMC. We found that the frequencies of OspA161–175-specific T cells declined during or soon after antibiotic therapy, months before the synovitis resolved in patients with antibiotic-refractory arthritis. This observation suggests that persistent synovitis in patients with refractory arthritis is not perpetuated by OspA161–175-specific T cells.


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

During the past 18 years we evaluated 134 patients with Lyme arthritis (age 12 or older) who received antibiotic therapy according to the guidelines now recommended by the Infectious Diseases Society of America (21). The study protocol, which included HLA typing and T cell analyses, was approved by the Human Investigations Committees at Tufts-New England Medical Center, Boston, MA (1987–2002) and Massachusetts General Hospital, Boston, MA (2002 to the present). All patients met the criteria of the Centers for Disease Control and Prevention for the diagnosis of Lyme disease (22, 23). As in previous studies (5), antibiotic-responsive arthritis was defined as the resolution of arthritis within 3 mo after no more than 4 wk of i.v. antibiotics or 8 wk of oral antibiotics, and antibiotic-refractory arthritis was defined as persistent joint swelling for ≥3 mo after the start of at least 4 wk of i.v. antibiotics, at least 8 wk of oral antibiotics, or both. PBMC and, if available, SFMC and serum samples were obtained at each visit and aliquots were frozen for subsequent determinations. The clinical characteristics, the results of treatment (5), and the HLA haplotypes and alleles (15) in most of these patients have been published previously. Of the 134 patients, 13 had the DRB1*0401 allele, and they are the subject of the current study.

Preparation of PBMC and SFMC

The mononuclear cells were separated from peripheral blood by Ficoll gradient centrifugation (lymphocyte separation medium; MP Biomedicals) as described previously (12). Synovial fluid was centrifuged directly to pellet the cells, which were washed three times with Dulbecco’s PBS (Invitrogen Life Technologies) by centrifugation at 1,200 rpm for 10 min. Both the PBMC and SFMC were frozen in aliquots of 5 x 106 cells/ml in 10% DMSO in FCS (Sigma-Aldrich) the same day of their collection and maintained in liquid nitrogen until ready for use.

Preparation of tetramers

The MHC class II tetramers were prepared at the Benaroya Research Center, Seattle, WA as previously described (24). Briefly, recombinant HLA-DR molecules were produced from chimeric cDNAs containing the extracellular domains of DRA1 and DRB1 molecules. The clones were expressed in S-2 Schneider cells and the HLA-DR molecules were purified by affinity chromatography. HLA-DRB1*0401 monomers (0.5 mg/ml) were biotinylated and loaded with the peptide of interest (5 mg/ml). In DRB1*0401-positive patients, the core immunodominant epitope of OspA is located at aa 165–173. In an effort to increase peptide-binding avidity, we included four peptide-flanking residues at the N-terminal end and two peptide flanking residues at the C-terminal end of the peptide (VLKSYVLEGTLTAEK), referred to here as OspA161–175. Two irrelevant control peptides were used. Initially, studies were done with influenza virus hemagglutinin (HA) aa 307–319 (HA307–319; PKYVKQNTLKLAT), but later the HSV2 viral protein 16 (VP16465–484; YGALDVDDFEFEQMFTDALG) was used for this purpose. Finally, loaded monomers were incubated with PE-conjugated streptavidin (Biosource International) to generate tetramers.

Tetramer staining

On the day of testing, frozen PBMC or SFMC were thawed and washed twice in PBS. The cells were stained according to a magnetic bead capture technique (25). First, 106 cells in 50 µl of PBS were stained with 2 µg/ml PE-labeled tetramers at room temperature for 2 h. During the last 20 min the cells were stained with 8 µl of anti-CD14 allophycocyanin, anti-CD19 allophycocyanin (BD Biosciences), and anti-CD4 FITC Abs at room temperature. The cells were washed at 1,000 rpm for 5 min at 4°C using the centrifuge’s low brake and stained with 20 µl of anti-PE beads (Miltenyi Biotech) at 4°C for 20 min. The cells were washed again and 10% of the cells (105 cells) were removed as the pre-enrichment fraction. The remaining 90% (9 x 105 cells) were passed through a MS separation column (Miltenyi Biotech) in 0.5 ml MACS buffer, which consisted of PBS (Invitrogen Life Technologies) with 0.5% BSA fraction V (Sigma-Aldrich) and 2 mM EDTA (Invitrogen Life Technologies). The unbound fraction was collected after washing the column three times with 0.5 ml of MACS buffer, and the eluate was collected in 2 ml of MACS buffer as the bound fraction.

After the three fractions were centrifuged, the cells were resuspended in 125 µl of FACS buffer and stained with 20 µl of ViaProbe (BD Biosciences) for 10 min before flow cytometric analysis. For flow cytometric analysis, all cells were acquired using a FACSCalibur device (BD Biosciences). All of the cells in each of the fractions were analyzed, including 5–8 x 104 cells in the pre-enrichment fraction, 4–7 x 105 cells in the unbound fraction, and 2–8 x 104 cells in the bound fraction. The lymphocytes were gated on forward scatter/side scatter, and the tetramer-PE+ cells were analyzed in the CD4+CD14–CD19–ViaProbe– lymphocyte population. The rationale for pregating CD4+CD14+ cells was to eliminate monocytes that bound the tetramers nonspecifically. The frequency of OspA161–175-specific T cells was calculated as the total number of tetramer PE+ cells in the bound fraction divided by the total number of CD4+ T cells in the unbound and bound fractions. Tetramer-PE-positive cells were not seen in the unbound fraction.

Proliferation assay

To confirm that T cells identified by the OspA-tetramer were Ag-reactive, initial samples of PBMC in which enough cells remained were tested in proliferation assays as previously described (26). Briefly, 5 x 105 cells in duplicate wells were stimulated with OspA161–175 (10 µg/ml) or no Ag. After 5 days in culture, the cells were pulsed with [3H]thymidine, and harvested 16–18 h later. The results were expressed as counts per minute in cells stimulated with the OspA peptide minus the counts with no Ag stimulation ({Delta}cpm).

ELISA

To correlate T and B cell results, serum samples obtained on the same date as PBMC were tested for anti-OspA IgG Abs by ELISA as previously described (26). Briefly, the plates were coated overnight with 5 µg/ml recombinant OspA. After the blocking and washing steps the plates were incubated with patients’ sera (1/50) followed by goat anti-human IgG conjugated with alkaline phosphatase (1/1,000; BioSource International). The substrate was para-nitrophenyl phosphate (Sigma-Aldrich). A standard curve was plotted from serial dilutions of a known positive control serum sample using Microsoft Excel (EFLAB program), and the titers of the unknown samples were extrapolated using the equation of the slope of the standard curve. The cutoff absorbance for a positive value was calculated as 3 SD above the mean absorbance of the eight normal serum samples tested in the same plate. Sera that had Ab levels above the linear part of the curve were diluted 2- to 8-fold more, and the ELISA was repeated until the Ab response fell in the linear section of the curve.

Statistics

OspA161–175-specific T cell frequencies were compared in patients with antibiotic-responsive and antibiotic-refractory arthritis using Student’s t test. Correlation between OspA161–175-specific T cell frequencies and anti-OspA IgG Ab responses was determined using linear regression coefficients (r values). The p values were calculated using the statistical calculator of VassarStats (Vassar College, Poughkeepsie, NY). All p values are two-tailed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Clinical description of the 13 DRB1*0401-positive patients

Of the 134 patients with Lyme arthritis seen during the past 18 years who were treated with Infectious Diseases Society of America-recommended courses of antibiotic therapy (21), 13 (10%) had the DRB1*0401 allele. Ten were men and three were women, and their median age was 25 (range: 12–64). Of the 13 patients, six had antibiotic-responsive arthritis. These patients, who were usually first seen before antibiotic therapy, received oral doxycycline for 1 or 2 mo and, by definition, their arthritis resolved within 3 mo after starting therapy. The seven patients with antibiotic-refractory arthritis were treated with oral doxycycline for 2 mo, ceftriaxone i.v. for 1 mo, or both, but they had persistent arthritis for a median duration of 9 mo (range: 5–48 mo) after starting therapy. These patients were usually referred because of lack of response to one or more courses of antibiotic therapy and, therefore, their initial samples were obtained a median duration of 2 mo after the initiation of antibiotics. After antibiotic treatment they were given anti-inflammatory medications, often including disease-modifying antirheumatic drugs, particularly hydroxychloroquine.

Cutoff value for the detection of OspA161–175-specific T cells

In initial experiments, PBMC from two DRB1*0401-positive normal control subjects and one DRB1*0401-positive patient with antibiotic-refractory Lyme arthritis were stained with the OspA161–175 tetramer and with two control tetramers, one containing a peptide from influenza virus HA (HA307–319) and the other containing a peptide from HSV type 2 (HSV2465–484). Both control subjects had low frequencies of CD4+ T cells enumerated with either the HA307–319 or HSV2465–484 tetramer (9 and 4 per 105 CD4+ T cells, respectively), and such cells were not identified in the Lyme arthritis patient (Fig. 1). Conversely, neither of the control subjects had cells enumerated with the OspA161–175 tetramer, whereas the Lyme arthritis patient had a high frequency of such cells (210 per 105 cells). We concluded that these tetramers could be used to assess all of the 31 patient samples (25 PBMC and 6 SFMC).


Figure 1
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  		FIGURE 1. Determination of tetramer staining characteristics using CD4+ T cells from two DRB1*0401-positive control subjects and one DRB1*0401-positive patient with Lyme arthritis by a magnetic bead capture technique. The column contained anti-PE-coated beads, and the streptavidin component of the tetramer-reagent was labeled with PE. Tetramer-positive cells were only found in the bound fraction. In control subject 1 the control tetramer was HA307–319 and it was HSV2465–484 in control subject 2 and in the patient. In the dot plots the percentage of tetramer-positive cells in the bound fraction is shown. However, the cell frequencies, which are given in the table on the right, were calculated based on the total CD4+ T cell population in the bound and unbound fractions.

 
With the first six samples tested the mean frequency of HA307–319 tetramer-positive cells was 4 ± 7 per 105 CD4+ T cells. Because Lyme arthritis patients were probably more likely to have had influenza vaccination or infection than herpes simplex infection, an HSV2465–484 tetramer was used in subsequent experiments. With the remaining 25 patient samples, the mean frequency of HSV2465–484 tetramer-positive cells was 1 ± 1 per 105 CD4+ T cells. However, because patient cells were limited, the first six experiments were not repeated with the HSV2465–484 tetramer. For this study, the detection limit of OspA161–175-specific T cells was defined as 3 SD above the mean value of the 25 patient samples using the HSV tetramer, which was 4 per 105 cells.

OspA161–175-specific T cell frequencies in PBMC in patients with Lyme arthritis

Among the 13 DRB1*0401-positive patients with Lyme arthritis, nine had single samples of PBMC or SFMC and four had serial samples. In initial samples of PBMC, three of the six patients (50%) with antibiotic-responsive arthritis and four of the seven patients (57%) with antibiotic-refractory arthritis had frequencies of OspA161–175-specific CD4+ T cells above the cutoff value of 4 per 105 cells (Table I). Among the six antibiotic-responsive patients, who were usually seen before antibiotic treatment, the mean frequency of OspA161–175-tetramer-positive cells in PBMC was 16 per 105 CD4+ T cells. Among the seven patients with antibiotic-refractory arthritis, who were first seen a median duration of 2 mo after the initiation of antibiotics, the mean frequency of OspA161–175-specific T cells in PBMC was 49 per 105 cells. Thus, antibiotic-responsive and antibiotic-refractory patients did not differ significantly in the number with positive results or in the frequency of OspA161–175-specific T cells.


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  		Table I. Frequency of tetramer-positive cells and proliferative response of PBMC in patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis

 
When PBMC from initial samples were stimulated with the OspA161–175 peptide in proliferation assays (5 x 105 cells were tested in each well), positive results (defined as ≥1,000 {Delta}cpm) were obtained in all seven patients who had frequencies of OspA161–175-specific T cells above the cutoff value of 4 per 105 cells, whereas negative proliferation results were found in the five patients in whom cell frequencies were below the cutoff value (Table I). However, among the seven patients with positive proliferation results the actual values did not correlate well with OspA161–175-specific T cell frequencies, presumably because of the random variation of the low numbers of OspA161–175-specific cells in individual wells or because of differences in the activation state of these cells.

OspA161–175-specific T cell frequencies in serial PBMC samples

In four patients, two with antibiotic-responsive arthritis and two with antibiotic-refractory arthritis, three or four serial samples were available. In all four patients the frequency of OspA161–175-specific CD4+ T cells declined with antibiotic therapy (Fig. 2). In the two patients with antibiotic-responsive arthritis (patients 1 and 4), the frequencies fell to levels near or below the cutoff value within 1–2 mo after starting treatment, when the arthritis resolved. In the two patients with antibiotic-refractory arthritis (patients 8 and 12), the highest values may have been missed because their initial samples were obtained several months after the initiation of antibiotic therapy. Even so, their OspA161–175-specific T cell frequencies in initial samples were higher than those in patients with antibiotic-responsive arthritis. In both patients the frequencies of OspA161–175-specific T cells declined to low or undetectable levels during the 2- to 3-mo period of antibiotic therapy (in patient 12 there was a gap of 4 mo between the start of the first and second courses of antibiotics). However, despite treatment with diclofenac and hydroxychloroquine during the postantibiotic period, their arthritis persisted for 11 or 18 mo after starting antibiotic therapy. Similarly, although only one sample was available in the remaining nine patients with responsive or refractory arthritis, only low or undetectable levels of OspA161–175-specific T cells were found in the four samples obtained 3 or more months after starting therapy.


Figure 2
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  		FIGURE 2. Correlation of the duration of joint inflammation and OspA161–175-specific T cell frequencies in serial samples from two patients (nos. 1 and 4) with antibiotic-responsive arthritis (A) and two patients (nos. 8 and 12) with antibiotic-refractory arthritis (B). In patient 12 there was a gap of 4 mo between the initiation of his first and second courses of antibiotics. The cross-hatched areas show the amount of joint swelling, and the lines with closed circles show the frequency of OspA161–175-specific T cells. In both antibiotic-responsive and antibiotic-refractory patients the frequencies of OspA161–175-specific T cells declined during the 1–3 mo period of antibiotic therapy, whereas joint inflammation persisted for months longer in the antibiotic-refractory cases.

 
OspA161–175-specific T cell frequencies in peripheral blood and synovial fluid

Concomitant PBMC and SFMC obtained during the infectious period were available in five patients, and a single SFMC sample obtained during the postantibiotic period was available in a sixth patient. Although synovial hypertrophy usually became more apparent in the postantibiotic period, joints were considerably less swollen by that time and, therefore, joint fluid was less often available.

Of the five patients with concomitant PBMC and SFMC, four (80%) had frequencies of OspA161–175-specific T cells above the cutoff value of 4 per 105 CD4+ T cells in both PBMC and SFMC (Fig. 3). In these four patients, the mean frequency of such cells in SFMC (476 per 105 cells) was 16 times higher than that in concomitant PBMC reaching levels as high as 1,177 per 105 cells (or ~1% of these cells). Patient 13, who had a single SFMC sample in the postantibiotic period, had a value of 6 cells per 105 cells, which was slightly above the cutoff value. Thus, during the infectious period, OspA161–175-specific T cells were concentrated in synovial fluid, the site of inflammation.


Figure 3
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  		FIGURE 3. Comparison of OspA161–175-specific T cell frequencies in PBMC and SFMC during the infectious period in two patients with antibiotic-responsive arthritis and three patients with antibiotic-refractory arthritis. In the final patient (patient 13), a single SFMC sample was available during the postantibiotic period. During the infectious period OspA161–175-specific T cells were concentrated in synovial fluid, but a smaller number of such cells were also found in peripheral blood. Control tetramers were used in every experiment, but the results, which were much lower with the control tetramers than with the OspA tetramer, are not shown.

 
Correlation of OspA161–175-specific T cell frequencies with anti-OspA IgG titers

Among 26 concomitant PBMC and serum samples there was a direct correlation between the number of OspA161–175-specific T cells and anti-OspA IgG titers (r = 0.4; p = 0.04) (Fig. 4). Similarly, in the four patients in whom serial samples were available, both the number of OspA161–175-specific T cells and the titers of anti-OspA Ab declined after antibiotic therapy, but the Ab responses fell more slowly than T cell numbers (data not shown). Among five concomitant SFMC and serum samples, OspA Ab titers also correlated well with OspA161–175-specific T cell frequencies (r = 0.6), but five values were not enough to show statistical significance. Because our previous experience is that Ab titers are similar in serum and joint fluid, we did not determine joint fluid Ab titers in these patients.


Figure 4
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  		FIGURE 4. Correlation of OspA161–175-specific T cell frequencies and anti-OspA IgG Ab titers in peripheral blood. The dotted lines denote the cutoff values for a positive response.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The enumeration of Ag-specific or autoreactive CD4+ T cells with tetramer reagents requires the detection of cells at a lower frequency and binding affinity than has usually been the case with CD8+ effector T cells (19). With the method used in our previous study (20), patients’ PBMC did not have frequencies of OspA164–175-specific CD4+ T cells that were higher than those found with a control tetramer. For this study, we used a longer OspA peptide (a 16-mer rather than a 12-mer); we loaded the peptide into empty DR binding grooves rather than linking it covalently, and we used a newer staining protocol. With these changes, the detection limit of OspA161–175-specific CD4+ T cells in PBMC was 4 per 105 cells. Other investigators have had similar or better results with these techniques in other diseases. With PBMC from patients with hepatitis C infection, the detection limit of Ag-specific CD4+ T cells was 0.1 per 105 cells (25). Additionally, in PBMC from healthy subjects who had been previously vaccinated for influenza, the frequencies of HA306–318-specific CD4+ T cells ranged from 0.6 to 3 per 105 cells (27), similar to the frequency of such cells in the six patient samples tested here. Thus, because of the low background frequency, it was possible to do a longitudinal analysis of OspA161–175-specific T cell frequencies in PBMC throughout the course of Lyme arthritis.

When the infection was active, ~50–60% of the DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory arthritis had increased numbers of OspA161–175-specific CD4+ T cells in peripheral blood and synovial fluid, and these Ag-specific cells were concentrated in joint fluid, the local site of inflammation. Within several months after starting antibiotic treatment, the frequencies of these cells declined to low or undetectable levels in both the responsive and refractory groups. These findings are completely consistent with the results of PCR testing for B. burgdorferi DNA in joint fluid (5, 6, 7), a surrogate marker for culture in Lyme arthritis. Patients with antibiotic-responsive arthritis often have positive PCR results before antibiotic therapy (5, 6). However, because joint inflammation resolves rapidly, joint fluid is usually not available for testing after starting treatment. In contrast, patients with antibiotic-refractory arthritis often have positive PCR results in joint fluid throughout the 1- to 3-mo period of antibiotic treatment, but the results are frequently negative after that time (5, 6, 7). Thus, patients with antibiotic-refractory Lyme arthritis probably remain infected for several weeks longer than those with antibiotic-responsive arthritis. However, even in patients with refractory arthritis, near or total spirochetal eradication seems to occur during the period of antibiotic treatment, months before the resolution of arthritis.

Our original plan was to determine OspA161–175-specific T cell frequencies not only in patients with the DRB1*0401 allele, but also in patients with other common antibiotic-refractory Lyme arthritis-associated DRB alleles, including the DRB1*0101 and DRB5*0101 alleles (15). However, even though samples from patients with the latter two alleles had T cells that proliferated in response to OspA161–175, tetramers with these same molecules did not seem to bind these T cells (data not shown). Previously, in vitro MHC/peptide binding assays showed that the DRB1*0401 molecule was the strongest binder of the OspA161–175 peptide (half maximal binding concentration = 0.003 µM), whereas the DRB1*0101 and DRB5*0101 molecules bound it only moderately well (0.3 or 0.4 µM) (15). Thus, the tetramer reagents used here seemed to require that DR molecules bind the peptide with high affinity (27).

Because of difficulties in enumerating OspA161–175-specific T cells in patients with DR molecules other than the DRB1*0401 molecule, we showed here that the frequencies of these T cells correlated with anti-OspA Ab titers, which can be determined easily in all patients. In a recent study (28), we determined the Ab responses to B. burgdorferi sonicates and four Borrelia Ags, including OspA, in 69 patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis. Consistent with the results in the present study, ~70% of the patients in both groups had Ab responses to OspA. These responses and those to the other spirochetal Ags declined gradually after antibiotic treatment (28), providing further evidence for the near of total eradication of spirochetes with antibiotic therapy. However, OspA titers tended to be higher in those patients with antibiotic-refractory arthritis than in those with antibiotic-responsive arthritis, whereas the titers to the other Borrelia Ags tested were quite similar in both patient groups. In the current study, there was a suggestion that patients with antibiotic-refractory arthritis may have higher frequencies of OspA161–175-specific T cells than those with antibiotic-responsive arthritis, but the differences were not statistically significant.

The current results provide insights regarding the pathogenesis of antibiotic-refractory Lyme arthritis. First, the decline in the frequencies of OspA161–175-specific T cells, the decrease in the Ab responses to multiple spirochetal Ags (28), and the negative PCR results for B. burgdorferi DNA in joint fluid (5, 6) argue against the hypothesis that persistent infection or retained OspA Ags perpetuate an inflammatory response in affected joints for months or even several years after antibiotic therapy. Rather, the triggering of infection-induced autoimmunity or a failure of immunoregulation seems more likely.

Human lymphocyte-associated Ag 1 (LFA-1{alpha}L332–340), a self-peptide having partial sequence homology with OspA165–173, was originally proposed as a candidate autoantigen in antibiotic-refractory Lyme arthritis (11). However, the LFA-1 peptide was later shown to act as only a weak partial agonist for OspA165–173–reactive DRB1*0401 T cells (29) and did not bind well to the refractory arthritis-associated DRB1*0101 molecule (16), making it unlikely to be a relevant autoantigen. In a recent search of the published human genome, no other human peptides that had sequence homology with OspA were identified that bound all antibiotic-refractory arthritis-associated DRB molecules and stimulated T cells in patients with antibiotic-refractory arthritis (30). The current study shows that some patients with an antibiotic-refractory course lacked T cell responses to OspA161–175 while others with antibiotic-responsive arthritis had such responses, and in both groups these responses declined with antibiotic treatment. Taken together, these results suggest that T cell reactivity with this OspA epitope, by itself or to a human molecular mimic of this epitope, would be an unlikely cause of antibiotic-refractory arthritis.

Other factors, such as particular chemokine and cytokine responses, must be required. We recently reported that patients with antibiotic-refractory arthritis had significantly higher levels of Th1 chemoattractants and cytokines in joint fluid, particularly CXCL9 and IFN-{gamma}, than antibiotic-responsive patients (31). Moreover, in patients with refractory arthritis these responses remained high or even increased in the postantibiotic period, even as cellular and humoral reactivity with borrelial proteins declined. These results and those in the current study seem to fit best with the postulate that OspA161–175 (or other currently unidentified borrelial epitopes) may cause exceptionally high inflammatory responses in genetically susceptible individuals. This may lead to bystander activation of autoreactive T cells, which may cause persistent joint inflammation, or this infection-induced inflammatory response may not be down-regulated appropriately.

In conclusion, ~50–60% of patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis initially had increased frequencies of OspA161–175-specific T cells. However, the marked decline in the frequency of such cells with antibiotic therapy in both patient groups suggests that persistent synovitis in the refractory group is not perpetuated by these cells.


   Acknowledgments
 
We thank Dr. Lee Ann Baxter-Lowe and David Sidebottom at the University of California, San Francisco, CA for performing HLA typing of patients, and Colleen Squires for help with preparation of the manuscript.


   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 in part by National Institutes of Health Grant AR-20358 and by the English-Bonter-Mitchell Foundation, the Eshe Fund, and the Lyme/Arthritis Research Fund at Massachusetts General Hospital, Boston, MA. P. K. received a scholarship for the study of Lyme disease from the Lillian B. Davey Foundation. Back

2 Address correspondence and reprint requests to Dr. Allen C. Steere, MD, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, 55 Fruit Street, Charlestown Navy Yard 149/8301, Boston, MA 02114. E-mail address: asteere{at}partners.org Back

3 Abbreviations used in this paper: OspA, outer surface protein A; HA, hemagglutinin; SFMC, synovial fluid mononuclear cell. Back

Received for publication May 8, 2007. Accepted for publication August 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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