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CD8 T Cells Are Not Essential to the Pathogenesis of Arthritis or Colitis in HLA-B27 Transgenic Rats¹

Harold C. Simmons Arthritis Research Center and Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390

	Abstract

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The class I MHC allele HLA-B27 is highly associated with the human spondyloarthropathies, but the basis for this association remains poorly understood. Transgenic rats with high expression of HLA-B27 develop a multisystem inflammatory disease that includes arthritis and colitis. To investigate whether CD8 T cells are needed in this disease, we depleted these cells in B27 transgenic rats before the onset of disease by adult thymectomy plus short-term anti-CD8 mAb treatment. This treatment induced profound, sustained depletion of CD8 T cells, but failed to suppress either colitis or arthritis. To address the role of CD8^+- cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8 mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8 regimen, or 4) no treatment. Arthritis occurred in 40% of each group, but was most significantly reduced in severity in the anti-CD8-treated group. In addition to CD8 T cells, two sizeable CD8^+- non-T cell populations were also reduced by the anti-CD8 treatment: 1) NK cells, and 2) a CD4⁺CD8⁺CD11b/c⁺CD161a⁺CD172a⁺ monocyte population that became expanded in diseased B27 transgenic rats. These data indicate that HLA-B27-retricted CD8⁺ T cells are unlikely to serve as effector cells in the transgenic rat model of HLA-B27-associated disease, in opposition to a commonly invoked hypothesis concerning the role of B27 in the spondyloarthropathies. The data also suggest that one or more populations of CD8^+- non-T cells may play a role in the arthritis that occurs in these rats.

	Introduction

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Over the past three decades, a number of genetic associations have been described between certain alleles or haplotypes of the human MHC and a variety of human diseases. Among these associations, one of the strongest and earliest to be described is the association of the class I allele HLA-B27 with the group of rheumatic disorders termed spondyloarthropathies. There is strong indirect evidence that the B27 molecule itself is a participant in the pathogenesis of these disorders (reviewed in Ref. 1). This evidence includes the association of disease with some, but not all, closely related subtypes of B27 distributed in human populations throughout the world; the absence of any strong association with genes genetically linked to the HLA-B locus; and the spontaneous development of arthritis and other clinical features of the spondyloarthropathies in rats and mice transgenic for HLA-B27.

Because the HLA-B27 allele is a typical MHC class Ia H chain, and because one of the principal functions of MHC class I molecules is to present peptide Ags to CD8⁺ CTL, it has long been hypothesized that CD8⁺ T cell recognition of B27 plays a role in the pathogenesis of the spondyloarthropathies (2). This hypothesis has prompted extensive searches for B27-restricted CD8⁺ T cells in patients with these disorders, and extensive characterization of the peptides bound to HLA-B27 that might be presented to these putative T cells (3, 4, 5). To date, these studies have been inconclusive, and no consensus has emerged regarding the role of CD8 T cells in B27-associated disease.

CD8 consists of two polypeptide chains, and , of the Ig superfamily (6). Cell surface-expressed CD8 exists as either heterodimers or homodimers. Thymus-derived CD8⁺ CTL generally express the CD8 heterodimer (7), and the binding of CD8 to MHC class I is thought to strengthen the Ag-specific binding of the TCR to the peptide/MHC class I complex (8). However, the CD8 homodimer is sufficient for binding to MHC class I (9, 10). It is therefore conceivable that HLA-B27 might interact with CD8 homodimers on one or more populations of cells in the course of disease pathogenesis.

More recently, it has become recognized that class I MHC molecules also interact with a large group of molecules on cell types other than thymus-derived CD8 T lymphocytes, and particularly with inhibitor and activation molecules on NK cells (11). Although this recognition is thought to be less dependent upon the particular peptides carried by individual MHC molecules, evidence exists for peptide-specific recognition of B27 by NK cells (12). Recognition of some form of B27 by CD4⁺ T cells has also recently been described (13). It has also been shown that B27 is somewhat unusual among MHC class I molecules in regard to the inefficiency of its biosynthesis (14, 15) and in its potential to form H chain dimers (15, 16), and it has been proposed that these features of B27 contribute to its propensity to predispose to rheumatic disease. Studies in mice transgenic for HLA-B27 with targeted deletions of ₂-microglobulin or TAP have also suggested that arthritis can occur in the absence of classical CD8⁺ T cell recognition of B27 (17). It thus remains possible that recognition of B27 by molecules other than Ag receptors on conventional CD8 T cells, and/or other aspects of B27 physiology and function, may provide the underlying basis for the extraordinary disease predisposition associated with HLA-B27.

Rats transgenic for HLA-B27 that express high levels of B27 mRNA and protein develop a spontaneous inflammatory disease that contains several features of the human spondyloarthropathies, including arthritis and colitis (18, 19). These disease manifestations are known to require T cells (20) and gut bacteria (21), and to be transferrable to nontransgenic rats by bone marrow transplantation (22). However, the role of specific T populations in this disorder has not been clarified. In experiments in which purified T cell populations were transferred to athymic rnn/rnu B27 transgenic rats, CD4 cells were more potent and consistently effective than CD8 cells in inducing gut inflammation (20), and the very low incidence of arthritis in any of the recipients prevented any conclusions to be drawn regarding this specific disease manifestation. A subsequent study suggested that the specificity of peptides bound to B27 influences the incidence of arthritis of B27 transgenic rats (23), but these data at best only indirectly implicate CD8 T cells, and they are also consistent with alternative explanations of the role of B27 that are not necessarily dependent on these cells.

In this work, we report the results of studies specifically aimed at determining whether CD8 cells play a critical role in the pathogenesis of the spontaneous inflammatory disease of HLA-B27 transgenic rats. The results strongly suggest that conventional CD8 T cells have no important role in this process, but that cells with other forms of CD8 may possibly participate in the pathogenesis of the arthritis seen in these rats.

	Materials and Methods

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Rats

Male rats of the disease-prone transgenic 21-4H or 33-3 lines carrying HLA-B27 and human ₂-microglobulin transgenes on the inbred LEW background (19) were bred and maintained in our colony, as previously described (18, 24). Nontransgenic littermates and LEW rats of the healthy transgenic lines 21-4L (HLA-B27) and 120-4 (HLA-B7) (19) served as controls in flow cytometry experiments.

Adult thymectomy

Thymectomies and sham thymectomies were conducted on 7- to 12-wk-old rats, as previously described (22).

Ab treatment in vivo

For depletion of CD8 T cells (25), thymectomized rats were treated with one of three regimens, described in Results, with dialyzed supernatant of hybridoma lines producing the anti-CD8 mAb OX8 or the isotype-matched control Ab TS2/18.1.1 (anti-human LFA-2 (26)). For depletion of all CD8-bearing cells, rats were injected i.p. twice weekly with OX8 as either 1 ml of dialyzed hybridoma supernatant or 50 µg affinity-purified Ab, beginning at age 5–13 wk and continuing for the duration of rat’s lifetime. Control rats were similarly treated with TS2/18.1.1 supernatant, or left untreated. Hybridoma cell cultures were grown in RPMI 1640 and 10% FCS plus standard additives, and affinity purification of mAb was obtained by acid elution from protein G-Sepharose. The concentration of OX8 Ab in separate batches of supernatant ranged from 15 to 25 µg/ml.

Clinical scoring

Rats were scored three times per week for arthritis (0–4 scale for each hind paw) and diarrhea (0–3 scale), as previously described (22). Rats with severe wasting or other signs of significant distress were euthanized.

Flow cytometry

Three- or four-color analysis of lymphoid cells was conducted on a FACSCalibur instrument (BD Biosciences, San Jose, CA) and analyzed with CellQuest 3.3 software. The mAb used were either directly conjugated with FITC, PE, or PerCP, or biotinylated and detected with APC-conjugated streptavidin. The following mAb were used (with corresponding Ags in parentheses): OX6 (MHC class II), OX8 (CD8), 341 (CD8), OX35 (CD4), 3.2.3 or 10/78 (CD161a, NKR-P1A), OX39 (CD25, IL-2R), OX40 (CD134), OX42 (CD11b/c), OX41 (CD172a), G4.18 (CD3), OX19 (CD5), OX33 (CD45RC), OX62 (integrin _M290), HRL1 (CD62L), R73 (TCR /), V65 (TCR /), and RP-1 (granulocyte-specific marker (27)). Specific references for these mAb are described previously (28). Conjugated mAb were either produced in our own laboratory by standard methods (FITC, biotin), or purchased from BD PharMingen (San Diego, CA).

Separation of T cell subpopulations

CD8⁺ and CD4⁺ subpopulations were negatively selected using the Vario MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) or the FACSorting system (BD Biosciences) using standard protocols.

Bromodeoxyuridine labeling

Rats were i.p. injected with 10 mg of bromodeoxyuridine (BrdU)⁵ in 1 ml of PBS and euthanized after 7 days. Intracellular staining of lymphoid cells was performed using a commercial kit according to the manufacturer’s recommendations (BrdU Flow Kit; BD PharMingen).

TCR spectratyping

TCR spectratyping was adapted from a previously reported procedure (29) using primers specific for rat TCRBV families 1–20 (30).

Statistics

Statistical analyses were made using the software Prism 2.0 (GraphPad Software, San Diego, CA).

	Results

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Permanent depletion of CD8 T cells has no significant influence on the prevalence or timing of clinical manifestations

In the absence of technology for targeted gene deletion in rats, we used the combination of adult thymectomy and treatment with the depleting anti-CD8 Ab OX8 to render B27 transgenic rats permanently depleted of CD8 T cells. Two separate protocols for administration of OX8 were used. Under the first protocol, OX8 or the isotype-matched control TS2/18.1.1 was given i.p. daily for 5 days beginning within 1 wk after thymectomy or sham thymectomy. Under the second protocol, two 5-day courses of daily i.p. injections were given, the first within 1 wk before, and the second starting 2 days after thymectomy or sham thymectomy. Rats were observed until both diarrhea and arthritis were seen or until age 6 mo, whichever came first.

A total of 23 rats underwent thymectomy and OX8 treatment (Table I, group A), and these were compared with 78 other rats in four control groups, including thymectomized rats treated with control Ab, sham-thymectomized rats treated with OX8 or control mAb or no Ab, and unmanipulated rats (Table I, groups B–E). The completeness of CD8 T cell depletion was confirmed by flow cytometry with the anti-CD8 mAb 341. All of the six rats in group A that were studied up to 3 mo after treatment with OX8 showed 0.6% CD8⁺ cells in peripheral blood, compared with 3–11% CD8⁺ cells among the 11 rats tested from the other four groups (0.4 ± 0.2% vs 6.6 ± 0.3%, p < 0.0001) (Fig. 1). There were no significant differences observed between the one- and two-course Ab treatment protocols, and the results for these rats are combined within each group in Table I.

View this table: [in this window] [in a new window]   Table I. Depletion of CD8 T cells has no significant influence on the prevalence or onset of clinical manifestations in HLA-B27 transgenic rats

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Thymectomy plus pulse OX8 treatment permanently depletes CD8 cells, but not CD8 cells. Expression of CD8 (A and C, mAb 341) and CD8 (B and D, mAb OX8) on PBMC from a 7-mo-old LEW.33-3 male rat thymectomized at age 2 mo, followed by daily OX8 for 5 days (A and B), or from a healthy line 21-4L male (C and D). Bold line in A represents staining for CD8; light line represents staining with control mAb. Percentage of positive cells: A, 0.6%; B, 20.7%; C, 11.1%; D, 16.1%.

To explore further the possibility that CD8⁺ T cells might play a role in the disease of the B27 transgenic rats, we examined these cells for markers of activation, regulation, and proliferation. Three-color staining of PBMC for CD8, CD4, and CD25 (IL-2R) demonstrated similar numbers of CD25⁺ CD4⁺ cells in diseased B27⁺ and healthy B27^- control rats (23.3 vs 25.8%), and also similar numbers of CD8⁺CD25⁺ cells (7.4 vs 8.5%). Expression of the T cell activation marker CD134 (OX40 Ag) was increased in the CD4 cells of the diseased B27 rats vs control (19.3 vs 6.9% in healthy control), in contrast to CD8⁺CD134⁺ cells, which were less numerous and not increased in diseased B27 rats vs control (1.7 vs 3.2%). In vivo incorporation showed almost negligible proliferative activity in the CD8⁺ cells of diseased rats (0.15% of CD8⁺BrdU⁺ cells), whereas 1.9% of the CD4⁺ cells were BrdU⁺.

We sought evidence for Ag-specific clonal proliferation of CD4⁺ and CD8⁺ T cells by TCRB complementarity-determining region 3 (CDR3) spectratyping, i.e., assessing the length of the CDR3 regions of TCR genes of sorted CD4⁺, CD8⁺, and CD4⁺CD8⁺ T cells from pooled popliteal, iliac, and brachial lymph nodes of diseased and control rats. Even rats up to 8 mo of age with severe arthritis and diarrhea showed CDR3 spectra with an evenly distributed band pattern, indicating completely polyclonal CD4⁺ and CD8⁺ T cell populations (data not shown). Similarly, CD4⁺CD8⁺ double-positive cells undergoing apoptosis (as determined by staining for annexin V) showed polyclonal TCRB CDR3 lengths (data not shown), thus providing no evidence for the existence of Ag-specific T cells that become activated and rapidly die. These data support the inference drawn above that CD8 T cells are not important in the disease of the B27 transgenic rats, and that the effect seen with CD8 depletion (vide infra) is exerted through other cell populations.

Continuous depletion of CD8⁺ cells attenuates arthritis

The experiments with thymectomized rats described above seemed to indicate that conventional CD8 T cells are not critical to the pathogenesis of the disease of B27 transgenic rats, and this tentative conclusion was supported by the experiments described in the previous section. However, because the development of diarrhea and arthritis was treated as endpoints in these experiments, many of the rats were euthanized as soon as both of these manifestations had appeared, and most of the other rats were euthanized at 6 mo of age. Thus, data on the severity of these manifestations and on overall survival were not obtained. Moreover, these experiments did not address the potential role of non-T cells or extrathymically derived cells that express CD8, but not CD8, which were not permanently depleted by the treatment regimen (Fig. 1B).

To address these issues, an additional group of rats underwent thymectomy and subsequent doses of OX8 to repeat the CD8 T cell depletion experiment described above to maintain depletion of all CD8⁺ cells. Three other groups of rats were studied as controls: one that was treated chronically with OX8, another that was treated chronically with control Ab, and one that was left untreated. In all four groups, the rats were observed until they died spontaneously or became moribund and were then euthanized, and the severity of arthritis and diarrhea was recorded three times weekly.

As shown in Table II, whereas the thymectomized group showed a modest, but statistically significant reduction in survival, the chronic anti-CD8 treatment with OX8 had no significant effect on survival, compared with the untreated rats. Somewhat surprisingly, the group treated with control Ab also showed a significant reduction in survival compared with the untreated controls, perhaps as a result of the antigenic load of chronic Ab administration, or an interaction of the TS2/18.1.1 mAb with rat cells that is below the level of detection by flow cytometry.

Anti-CD8 treatment has no effect on inflammatory bowel disease

None of the treatments had any clinically evident effect on the spontaneous inflammatory bowel disease seen in the B27 transgenic rats. There were no significant differences among the four groups in the prevalence or age of onset of diarrhea, the age when maximal diarrhea was first seen, or the mean peak diarrhea score (data not shown).

Effects of anti-CD8 treatment on cell populations

Flow cytometry was conducted on peripheral blood cells from rats treated with CD8-depleting or control regimens. In group H, the group of rats treated with thymectomy and three subsequent injections of OX8 on consecutive days, 12 of the 14 rats had <0.5% CD8 cells in peripheral blood when analyzed 15–26 days after thymectomy, compared with 10% CD8 cells in control rats, indicating that this depletion regimen was as effective as the more intensive regimens used in group A. The other two rats in group H had 1.3 and 4.5% CD8 cells, indicating incomplete thymectomy and/or incomplete OX8-induced depletion; the former of these two rats developed arthritis, whereas the latter did not.

The depletion of the entire population of CD8⁺ cells by chronic administration of OX8 was not as complete as the specific depletion of thymic-dependent CD8 T cells by thymectomy and pulse OX8 treatment. Nonetheless, there was a significant reduction of 2-fold in the number of CD8⁺ cells, compared with the rats treated with control mAb (Table IV). Among the OX8-treated rats, there was no correlation between the arthritis score and the percentage of CD8⁺ cells (p = 0.74), nor was the percentage of CD8⁺ cells in the rats with arthritis significantly different from the percentage in the rats without arthritis (p = 0.73).

View this table: [in this window] [in a new window]   Table IV. Treatment with anti-CD8 significantly depletes CD8+ cells

In addition to conventional CD8 T cells, at least four other populations of CD8⁺ cells could be identified in peripheral blood and spleen, including NK cells, a macrophage population, and CD8^+- T cells. The NK cells are identified by bright staining for the NK marker CD161a (NKR-P1A) with the mAb 3.2.3 or 10/78 (not shown), and intermediate to high levels of expression of CD8 (Fig. 2, A and B). A similar, but distinct population with intermediate levels of NKR-P1A and CD11b/c and variable levels of CD8 was also commonly found (not shown). In healthy control rats, the macrophage population is largely negative for OX8, and is identified by the mAb (and corresponding markers) OX35 (CD4), OX42 (CD11b/c), 3.2.3 (CD161a), and OX41 (CD172a), and by the absence of staining for the granulocyte marker RP-1 (Fig. 2A, data for CD172a and RP-1 not shown). This population consistently expands early in the course of the spontaneous inflammatory disease in the B27 transgenic rats (Fig. 2, compare A and B). Chronic treatment with anti-CD8 reduced all of these populations (Fig. 2C), although to varying degrees from rat to rat.

View larger version (58K): [in this window] [in a new window]   FIGURE 2. Representative flow cytometry data, showing the effect of disease and of anti-CD8 treatment on CD8+ cell populations in peripheral blood. A, Three-month-old healthy control rat. B, Three-month-old rat of the 21-4H line with grade 3 diarrhea. C, Three-month-old rat of the 21-4H line treated for 27 days with OX8 mAb. Staining was with PerCP-OX8 (CD8) and PE-OX35 (CD4) in A and B, and with FITC-OX8 and PE-OX35 in C. The regions, corresponding cell populations, and respective statistics for the three panels are given in Table V. The histograms at left show the staining of cells from the indicated regions, stained for CD11b/c (FITC-OX42) and OX161a (biotinylated-3.2.3 + APC-streptavidin). Staining with negative control mAb is shown in the panel for CD11b/c from Aa. Mean channel fluorescence values, top to bottom, for CD11b/c are 58, 99, 113, and for CD161a are 43, 85, and 99, respectively. Rats A and B were analyzed together in the same experiment, along with three similar healthy controls and three similar diseased B27 transgenic rats. Similar results were obtained for all four rats within each group, represented by A and B.

In untreated rats, 10–15% of the CD8^+- cells were positive for CD3, CD5, and/or the TCR , and 1–5% of the CD8⁺⁺ cells were also CD4⁺. The effect of the interventions on these minor T cell populations was not studied systematically. Cells expressing the TCR were virtually undetectable (not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8 T cells do not influence the course of inflammatory disease in B27 transgenic rats

Recent evidence suggests that autoreactive CD8 T cells play an important effector role in the pathogenesis of murine models of type 1 diabetes mellitus, and potentially also in human type 1 diabetes, and similar evidence is accumulating for murine experimental autoimmune encephalomyelitis and human multiple sclerosis (reviewed in Ref. 31), despite the fact that these diseases are classically associated with specific MHC class II alleles. In contrast, in human ankylosing spondylitis and in the HLA-B27 transgenic rat model, it has been difficult to establish a role for CD8 T cells, despite the predominate importance of the MHC class I B27 molecule in these disease processes. In this communication, we report that depletion of CD8 T cells in still healthy young adult B27 transgenic rats of the disease-prone lines had no ameliorating effect on subsequent disease development and progression. Moreover, there was a lack of evidence for activation or expansion of a population of CD8 T cells in B27 transgenic rats with characteristic inflammatory disease.

These results are counter to the concept that presentation of one or more peptide Ags by HLA-B27 to CD8 T effector cells plays a role in the pathogenesis of inflammatory disease in the B27 transgenic rats. The results do not entirely exclude the possibility that the degree of depletion of CD8 T cells was insufficient to prevent the critical role of these cells, but the failure to achieve any significant disease amelioration and the persistence of the profound depletion make this unlikely. The results also do not entirely exclude the possibility that CD8 T cell recognition of B27 might play a critical, but indirect role before onset of clinically evident disease, for example during development of the T cell repertoire, but they argue strongly against a role for such cells as effector cells in the rat spondyloarthropathy model. We have previously observed in radiation bone marrow chimeras that the disease of the B27 transgenic rats can arise in the absence of any thymic exposure to B27 (20), and the present results indicating the lack of any need for B27-restricted CD8⁺ cells are consistent with this finding.

Provisional evidence that CD8^+- cells influence the severity of arthritis in B27 transgenic rats

In contrast to the lack of effect of the thymectomy plus pulse anti-CD8 mAb treatment, chronic treatment with the anti-CD8 mAb OX8 significantly reduced the duration and severity of arthritis in the B27 transgenic rats. This result suggests that one or more populations of CD8-expressing cells that are not affected by the thymectomy intervention serve as effector cells in the pathogenesis of arthritis in these rats. Although the rats treated with an isotype control Ab also showed a significant reduction in arthritis severity compared with untreated controls, there was a further significant reduction in the OX8-treated rats. Moreover, the rats treated with control Ab showed an accelerated mortality compared with untreated controls, suggesting that the apparent effect on arthritis severity in this group might be at least partly explained by the statistical artifacts of right truncation or censoring (32). Although further investigation will be needed to fully clarify the specificity of the chronic OX8 treatment, these results nonetheless illustrate that arthritis severity in the B27 transgenic rats is susceptible to amelioration by cell depletion protocols, and thus serve as a positive control for the experiments in which specific depletion of CD8 T cells had no effect.

Several sizeable CD8^+- populations were readily detected in peripheral blood and spleen, including NK cells and cells with a monocyte-macrophage phenotype that expanded early in the course of the disease. Both of these populations were diminished by chronic OX8 administration. In the case of NK cells, concurrent disappearance of staining for the mAb 3.2.3 on peripheral blood and spleen cells confirmed that the cells were actually eliminated. In the case of the monocytes, the persistence of CD8^- cells with an otherwise similar phenotype leaves open the possibility that the cells persist with down-regulated CD8 expression.

Intraepithelial lymphocytes (IEL) are another major population of cells expressing CD8. In rats, intestinal IEL predominantly express CD3 and the TCR , with CD4⁺CD8^+-, CD4^-CD8^+-, and CD4^-CD8⁺⁺ phenotypes all being common (33, 34). In this study, we did not examine intestines for the effects of the anti-CD8 interventions on IEL. However, any effect of these interventions on IEL would have occurred without any clinically evident impact on intestinal inflammation, which showed no variation among the different groups of rats in our study.

Two recent reports have focused on the population CD4⁺CD8⁺ peripheral T cells in rats (35, 36). This population contains both CD8⁺⁺ and CD8^+- phenotypes, both of which express predominantly TCR and which decline in frequency with age. Somewhat surprisingly, both the CD8⁺ and CD8^- populations appeared thymus dependent, and both were concluded to be recent thymic emigrants in transition to becoming CD4⁺ peripheral T cells. Although we did not systematically look for these populations, we observed that a small proportion of CD8^+- cells was positive for T cell markers, and that a very small proportion of CD8⁺⁺ cells was positive for CD4, results that are consistent with these recent reports.

Further investigation will be needed to identify which of these CD8⁺ cell populations contributes to arthritis in the B27 rats, and by what mechanism. Cell surface-expressed CD8 is thought to exist as either heterodimers or homodimers. Recent attention has been focused on NK cells and their manifold receptors in the spondyloarthropathies (37, 38), and the potential role of these cells in the B27 transgenic rats clearly merits further investigation. The CD8⁺ monocyte population is also of great potential interest. We previously reported an accumulation of OX42 (CD11b/c)⁺ cells in nontransgenic rats in which inflammatory disease was induced by transplantation of bone marrow cells from the disease-prone B27 transgenic lines (22), and the OX42⁺ cells observed in this study presumably reflect this same population. These cells seem rather definitely to be of a monocyte/macrophage lineage, because they express the myelomonocytic markers CD11b/c and CD172a and lack the myeloid marker RP-1 and the dendritic cell marker OX62. However, their phenotype is somewhat different from that reported for other inflammation-associated rat monocyte/macrophage populations (39, 40), because they do not show up-regulated MHC class II or CD62L, compared with control rats (unpublished data).

CD8 T cells may play a role in reactive arthritis

Whatever modest success that has been gained from efforts to identify B27-restricted CD8 T cells in the spondyloarthropathies has come largely from studies of reactive arthritis (3, 41). It is noteworthy that a shared TCR variable gene sequence has been found in clones isolated in different laboratories from synovial fluid from different patients with reactive arthritis triggered by different infectious agents, whereas clones from other forms of spondyloarthropathy did not carry this sequence (42, 43). It may thus be that the role of B27 in the pathogenesis of reactive arthritis is distinct from its role in the other spondyloarthropathies. As described in this work, we have been unable to identify T cell oligoclonality associated with early disease in the B27 transgenic rats. Taken together, the results suggest that the pathogenesis of spontaneous disease in the B27 transgenic rats may resemble other forms of spondyloarthropathy more closely than reactive arthritis.

In conclusion, the results presented in this study provide indirect support for several recent lines of investigation based on the concept that the role of HLA-B27 in most forms of spondyloarthropathy, including the B27 transgenic rat model, probably involves unusual behavior on the part of B27 that is distinct from the classical function as a restriction element for CD8⁺ T cells, and that may predominately involve innate immunity (44, 45).

	Footnotes

² Current address: Department of Biologie II, Ludwig-Maximilians-Universität, 80333 Munich, Germany.

³ Current address: Department of Medicine, University of Indiana, Indianapolis, IN 46202.

⁴ Address correspondence and reprint requests to Dr. Joel D. Taurog, Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8884. E-mail address: joel.taurog{at}utsouthwestern.edu

⁵ Abbreviations used in this paper: BrdU, bromodeoxyuridine; CDR3, complementarity-determining region 3; IEL, intraepithelial lymphocyte.

Received for publication August 9, 2002. Accepted for publication November 5, 2002.

	References

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