Genetic Regulation of T Regulatory, CD4, and CD8 Cell Numbers by the Arthritis Severity Loci Cia5a, Cia5d, and the MHC/Cia1 in the Rat

Abstract

T cells have a central role in the pathogenesis of autoimmune arthritis, and several abnormalities in T cell homeostasis have been described in rheumatoid arthritis (RA). We hypothesized that T cell phenotypes, including frequencies of different subsets of T regulatory (Treg) cells and in vitro functional responses could be genetically determined. Furthermore, we considered that the genetic contribution would be accounted for by one of the arthritis regulatory quantitative trait loci (QTL), thus providing novel clues to gene mode of action. T cells were isolated from thymus, peripheral blood, and spleen from DA (arthritis-susceptible) and ACI and F344 (arthritis-resistant) strains and from F344.DA(Cia1), DA.F344(Cia5a), and DA.F344(Cia5d) rats congenic for arthritis QTL. T cell subpopulations differed significantly between DA, F344, and ACI. DA rats had an increased frequency of CD4⁺ cells, and a reduction in CD8⁺ and CD4⁺CD45RC^|o Treg cells, compared with F344. The differences in CD4/CD8 and CD4⁺CD45RC^|o Treg cells were accounted for by Cia5a. DA rats also had a reduced frequency of CD8⁺CD45RC^|o CD25⁺ Treg cells compared with F344, and that difference was explained by Cia5d. DA rats also had a significantly lower frequency of CD4⁺CD25⁺ and CD8⁺CD25⁺ thymocytes, and of peripheral blood CD8⁺CD45RC^|o Treg cells, compared with F344 rats, and that difference was accounted for by the MHC. This is the first identification of arthritis severity QTL regulating numbers of CD4⁺CD45RC^|o (Cia5a) and CD8⁺CD45RC^|o CD25⁺ (Cia5d) Treg cells. The MHC effect on CD8⁺ Treg cells and CD25⁺ thymocytes raises a novel potential explanation for its association with arthritis.

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting approximately 1% of the population. Available treatments induce significant clinical improvement, but rarely achieve cure or remission (1). RA has a strong genetic component, with a heritability of 60% (2), and therefore the identification of genes contributing to disease susceptibility and severity is expected to generate novel and better therapeutic targets. Indeed, several genome-wide studies have identified MHC and non-MHC susceptibility loci (3–7), but little is known about the identity and mode of action of those genes, or of those regulating arthritis severity.

Several arthritis severity quantitative trait loci (QTL) were identified in intercrosses between arthritis-susceptible DA and arthritis-resistant F344 or ACI rats (8–10), and we confirmed the arthritis regulatory effect in congenic strains (11–15). Once identified, the arthritis genes contained within those QTL congenic intervals will elucidate pathways and processes central to disease pathogenesis. We also considered the possibility that immunologic phenotypes known to be relevant to the pathogenesis of arthritis, such as T cell phenotypes, may be genetically regulated, thus generating early clues to candidate QTL genes or pathways for further characterization.

T cells are known to have a central role in the pathogenesis of pristane-induced arthritis (PIA) (16,17), collagen-induced arthritis (CIA) (18–21), and RA (22). Early synovial T cell infiltrates in rodents contain increased numbers of CD4⁺ cells (23), and depletion of these cells prevents (24) and treats established disease (25). Additionally, CD4⁺ T cells contribute to autoantibody production, inflammation, synovial angiogenesis, hyperplasia, and cartilage and bone destruction in PIA, CIA, and RA (26–31), and T regulatory (Treg) cells are implicated in the suppression of cellular and humoral responses in CIA (32–34) and RA (35). Furthermore, abnormalities in T cell homeostasis and in in vitro responses have been described in RA (36,37) and may be genetically determined.

We specifically hypothesized that the rat chromosome 10 QTL Cia5a and Cia5d, which determine PIA and CIA severity, may contribute to the genetic regulation of T cell phenotypes. Cia5a determines levels of autoantibodies against cartilage type II collagen, and similarly to Cia5d regulates synovial events involved in disease pathogenesis, such as infiltration with mononuclear cells, production of IL-1β, angiogenesis, hyperplasia, and bone and cartilage erosions (14), thus making both the Cia5a and Cia5d QTL obvious candidates for T cell analyses.

In the present study, we demonstrated for the first time that the arthritis-susceptible DA differs form the arthritis-resistant ACI and F344 rat strains in several T cell phenotypes, indicating genetic regulation, and that genes controlling T cell numbers, including subsets of Treg cells, co-segregate with the arthritis severity loci Cia5a, Cia5d, and the MHC (Cia1), providing novel insight into the mode of action of these arthritis genes.

MATERIALS AND METHODS

Rats and Construction of the QTL Congenic and Subcongenic Lines

Specific pathogen-free 8- to 12-week-old female DA/Hsd (DA, arthritis-susceptible) and F344 and ACI (both arthritis-resistant) rats were purchased from Harlan-Sprague Dawley (Indianapolis, IN, USA), and DA/BklArb rats were purchased from Bentin & Kingman (Freemont, CA, USA). The rats were maintained at the Arthritis and Rheumatism Branch at the National Institutes of Health in Bethesda (Arb, NIAMS, NIH) and subsequently transferred to the Feinstein Institute for Medical Research (FIMR). Construction of the DA.F344(Cia5a) and DA.F344(Cia5d) subcongenic and F344.DA(Cia1) congenic lines was previously described (11,12,14). Briefly, an 89.7-Mb interval containing the 2-LOD support interval comprising Cia5 was introgressed from F344 into DA/BklArb rats through eight genotype-guided backcrosses, followed by intercrossing to generate homozygous DA.F344(Cia5) congenic rats. These congenics were further backcrossed twice to generate recombinants at the Cia5a (26.4 Mb) and Cia5d (47.3 Mb) intervals. Identical recombinants were intercrossed to generate DA.F344(Cia5a) and DA.F344(Cia5d) homozygous subcongenic lines (Figure 1). Similarly, the 32.6-Mb 2-LOD interval containing Cia1, which includes the rat MHC, was introgressed from DA/BklArb into F344 rats through eight genotype-guided back-crosses followed by at least five intercrosses to generate homozygous F344.DA(Cia1) congenic rats.

Figure 1.

Rat chromosomes 10 and 20 microsatelite (SSLP) markers used in the breeding of DA.F344(Cia5a), DA.F344(Cia5d), and F344.DA(Cia1) congenic strains. Numbers indicate interval distance in megabases (Mb). White: homozygous DA alleles; grey: region of recombination; black: homozygous F344 alleles. Physical positions were retrieved from Ensembl Rat (http://www.ensembl.org/Rattus_norvegicus/) according to the Rat Genome Assembly version RGSC 3.4.

All rats were housed in cages with two to three animals, in 12-h light cycles, and received water and regular chow ad libitum. All animal experiments were reviewed and approved by the FIMR Institutional Animal Care and Use Committee.

T cell isolation

Peripheral blood, spleen, and thymus were obtained from 8- to 12-week-old nonimmunized rats. Spleen tissue was teased and digested for 30 min with collagenase D (Roche, Indianapolis, IN, USA), and DNase I (Sigma-Aldrich, St. Louis, MO, USA). Mononuclear cell suspensions were isolated through density gradient separation with Lymphocyte Separation Medium (Mediatech, Herndon, VA, USA), and depleted of residual erythrocytes with Gey’s lysis buffer. Peripheral blood, thymus, and spleen mononuclear cell suspensions were selectively depleted of B, NK, and myeloid cells with magnetic beads coated with antibodies against OX43, CD172a (SIRP), CD161, and CD45RA (StemCell Technologies, Vancouver, BC, Canada), resulting in a purified suspension of negatively selected T cells (99% CD3⁺ in blood and spleen) and thymocytes (99% CD90⁺ in thymus). T cells were stained with Turk’s solution and counted. T cells and thymocytes were 93% to 97% viable based on trypan blue (Cambrex, Walkersville, MD, USA) staining.

Flow-cytometric characterization of T cell subsets

Freshly obtained T cells were suspended in PBS with 0.02% azide (Sigma-Aldrich) and 1% bovine serum albumin (P Biomedicals, Aurora, OH, USA), then incubated with anti-CD32 (BD-Pharmingen, San Diego, CA, USA) to block FcγII receptors, and four-color stained with saturating concentrations of different combinations of FITC, PE, PerCP, APC, or biotin-conjugated monoclonal antibodies to CD4 (Serotec, Raleigh, NC, USA), CD3, CD8, CD90, CD45RC, and CD25 (BD-Pharmingen), or the respective isotype controls. Stained cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry within 72 h in a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), using the BD Cell-Quest Pro version 4.0.1 software (Becton Dickinson).

In vitro T cell stimulation, activation, proliferation, and activation-induced cell death (AICD)

T cells were plated in 96-well plates at 5 × 10⁵ cells per well (200 μL/well), stimulated with plate-bound anti-CD3 (0.75 μg/well, BD-Pharmingen) plus anti-CD28 (2 μg/mL, BD-Pharmingen), and cultured in RPMI 1640 (Cell-gro, Herndon, VA, USA) with 10% fetal bovine serum (Mediatech), 2 mM glutamine, 20 μg/mL gentamicin, 0.5 μg/mL amphotericin B (Invitrogen, Carlsbad, CA, USA), and 50 μM β2-mercaptoethanol (Sigma-Aldrich) at 37°C and 5% CO₂.

T cell activation

Cell surface expression of CD25 (flow cytometry) was used to determine the frequency of activated cells before and 24 h after in vitro stimulation.

T cell proliferation

T cells were labeled with 1 μM carboxy-succinimidyl-fluoresceinester (CFSE, BD-Pharmingen) and washed with PBS. CFSE incorporation was confirmed by flow cytometry prior to plating. CFSE intensity was measured again 72 h after in vitro stimulation, and the proliferative index was calculated with ModFit LT version 2.0 (Verity Software House, Topsham, ME, USA). Specifically, the proliferative index indicates the average number of cell divisions that the original population underwent, and it is determined by the sum of cells in all generations divided by the calculated number of total precursor cells in the original population (38).

AICD

AICD at 24, 72, and 96 h of in vitro stimulation was quantified by flow cytometry, and cells were considered viable when stained negative for both annexin-V and 7-aminoactinomycin (7-AAD, BD-Pharmingen).

Quantification of cytokines

Supernatant aliquots were collected from splenic T cell cultures after 72 h of stimulation and stored at −80°C until used. Commercially available ELISA kits were used for the determination of IFNγ, TNFα, IL-4, and IL-10 (R&D, Minneapolis, MN, USA). All assays were run in duplicates.

Statistical Analysis

Medians were compared with the Mann-Whitney U test. Differences in cytokine absorbance values were normally distributed, and were compared with the Student t test. SigmaStat version 3.0 (SPSS) was used for statistical analyses. A P value of 0.05 was considered significant.

RESULTS

Cia5a accounts for the difference in percentage of CD4 and CD8 T cells seen in DA and F344 rats

The frequency of mature peripheral blood and spleen CD4 T cells (CD4⁺CD90⁻) was significantly higher in DA compared with F344 rats (P ≤ 0.001, Figure 2A; and P = .008, Figure 2B, respectively), suggesting that numbers of CD4⁺ cells are genetically regulated. That difference was not explained by the MHC, as F344.DA(Cia1) congenics had CD4⁺ cell numbers similar to F344. Instead, the higher number of CD4⁺ cells in DA was accounted for by the non-MHC locus Cia5a, as the number of CD4⁺ CD90⁻ cells in DA.F344(Cia5a) rats was similar to F344 (P = .002, Figure 2A; P = .033, Figure 2B).

Figure 2.

Frequency of CD4⁺ and CD8⁺ mature T cells and recent thymic emigrants (RTE). Peripheral blood and spleen of F344 and F344.DA(Cia1) rats had decreased frequency of CD4⁺ mature T cells (A–B) and CD4⁺ RTEs (C–D). Cia5a accounted for the circulating levels of CD4⁺ T cells in blood (A) and spleen (B), and of CD4⁺ RTE in blood. Peripheral blood and spleen of F344 and F344.DA(Cia1) rats had increased frequency of CD8⁺ T cells (E–F) and RTE (G–H). Cia5a accounted for the difference in mature CD8⁺ T cells (E–F), but not RTE (G–H). Results are expressed as medians ± 25–75 percentiles for ACI (n = 9), DA (n = 9), F344 (n = 9), DA.F344(Cia5a) (n = 5), DA.F344(Cia5d) (n = 6) and F344.DA(Cia1) (n = 6) rats. All results were compared with DA. **P ≤ 0.01; * P ≤ 0.05; † P = .052; Mann-Whitney U test.

Peripheral blood and spleen CD4⁺ recent thymic emigrants (RTE, CD4⁺ CD90⁺) followed a distribution similar to CD4⁺ mature T cells, with increased percentages in DA compared with F344 (P = .004, Figure 2C; and P = .013, Figure 2D, respectively), and this difference was not explained by the MHC. The difference in the frequency of CD4⁺CD90⁺ RTE cells was explained by Cia5a in peripheral blood (P = .018, Figure 2C), but not in the spleen (Figure 2D). These observations indicate that Cia5a accounts for the variation in frequency of mature and RTE CD4⁺ T cells, with the exception of CD4⁺ RTE in spleen, suggesting that early migration of RTE to the spleen is regulated by genes outside the Cia5a interval.

Conversely, peripheral blood and spleen from DA rats had significantly lower frequencies of both CD8⁺ CD90⁻ (P ≤ 0.001, Figure 2E; P = .006, Figure 2F, respectively) and CD8⁺ CD90⁺ T cells compared with F344 rats (P ≤ 0.001, Figure 2G; P = .003, Figure 2H). The non-MHC locus Cia5a accounted for the difference in the percentage of mature CD8⁺ CD90⁻cells (P = .002, Figure 2E; P = .016, Figure 2F), but not the difference of CD8⁺ CD90⁺ RTE (Figure 2G–H), detected in peripheral blood and spleen of DA and F344 rats. These observations implicate Cia5a in the regulation of levels of CD8⁺ CD90⁻T cells and suggest that a non-MHC gene other than Cia5a or Cia5d regulates levels of CD8⁺ CD90⁺ cells.

The number of mature splenic CD4⁺ and CD8⁺ T cells in DA.F344(Cia5d) were similar to F344 but different from DA rats [DA.F344(Cia5d) versus DA: CD4, P = .052, Figure 2B; CD8, P = .068, Figure 2F], suggesting that this locus may also regulate differences between DA and F344. There was no significant difference in the number of CD4⁺ and CD8⁺ T cells between DA and ACI rats.

The MHC regulates the frequency of thymocytes with a CD25⁺ regulatory phenotype

F344 rats had a 2.4-fold increased frequency of CD4⁺ CD25⁺ thymocytes (P = .034, Figure 3A), and a fourfold increased frequency of CD8⁺ CD25⁺ thymocytes (P = .004, Figure 3B) compared with DA. F344.DA(Cia1) and DA rats had similar frequencies of CD4⁺ CD25⁺ and CD8⁺ CD25⁺ thymocytes, demonstrating that the MHC accounts for the difference between DA and F344 rats. The numbers of CD4⁺ CD25⁺ and CD8⁺ CD25⁺ thymocytes were also similar in ACI and DA rats, which have the same MHC haplotype, further supporting the MHC contribution. The frequency of CD4⁺ CD25⁺ and CD8⁺ CD25⁺ T cells in peripheral blood and spleen of DA, F344, and ACI rats was not significantly different (data not shown).

Figure 3.

Frequency of CD25 surface expression in thymocytes. F344 rats had increased frequency of (A) CD4⁺ CD25⁺ and (B) CD8⁺ CD25⁺ thymocytes. DA and F344.DA(Cia1) rats had similar frequencies of CD4⁺ CD25⁺ and CD8⁺ CD25⁺ thymocytes. Results are expressed as medians ± 25–75 percentiles for ACI (n = 9), DA (n = 9), F344 (n = 9), and F344.DA(Cia1) (n = 6) rats. All results were compared with DA. ** P ≤ 0.01; * P ≤ 0.05; Mann-Whitney U test.

Cia5a regulates the frequency of CD4⁺ CD45RC^|o regulatory cells

The frequency of CD4⁺ CD45RC^|o cells in peripheral blood and in the spleen was increased in F344 rats (P ≤ 0.001, P = .021, respectively) and F344.DA(Cia1) rats (P = .001, P = .058, respectively) compared with DA rats (Figure 4A–B), indicating that it is predominantly influenced by genes outside the MHC. The role of non-MHC genes in the regulation of the number of CD4⁺ CD45RC^|o T cells was further demonstrated by the significant differences between ACI and DA rats (peripheral blood: P = .002, Figure 4A; spleen: P = .038, Figure 4B). The percentage of CD4⁺ CD45RC^|o T cells in peripheral blood of DA.F344(Cia5a) rats was similar to F344 and different from DA (P = .006, Figure 4A), indicating regulation by Cia5a. The percentage of CD4⁺ CD45RC^|o T cells in DA.F344(Cia5d) congenics was similar to DA (Figure 4A–B), demonstrating that F344 alleles at the Cia5d locus are not involved in the regulation of the number of these cells.

Figure 4.

Frequency of CD45RC^|o T cells. The frequency of CD4⁺ CD45RC^|o T cells was increased in F344 and F344.DA(Cia1) in both peripheral blood (A) and spleen (B). Cia5a accounted for the difference of CD4⁺ CD45RC^|o T cells in peripheral blood (A). ACI rats had reduced frequency of CD4⁺ CD45RC^|o (A–B). CD8⁺ CD45RC^|o T cells were also increased in F344 peripheral blood (C), but not in spleen (D), which was not observed in F344.DA(Cia1) rats (C). Results are expressed as medians ± 25–75 percentiles for ACI (n = 10), DA (n = 10), F344 (n = 10), DA.F344(Cia5a) (n = 5), DA.F344(Cia5d) (n = 9) and F344.DA(Cia1) (n = 6) rats. Compared with DA: **P ≤ 0.01, *P ≤ 0.05, †P = .058. Compared with F344: ‡P = .039. Mann-Whitney U test.

The MHC regulates the frequency of CD8⁺ CD45RC^|o regulatory cells

The frequency of peripheral blood CD8⁺ CD45RC^|o T cells was also higher in F344 compared with DA rats (P = .031, Figure 4C), but F344.DA(Cia1) had a frequency similar to DA, indicating that genes within the MHC account for this difference. Accordingly, there was no difference in the number of CD8⁺ CD45RC^|o T cells between DA and ACI rats. Cia5a and Cia5d did not influence the numbers of CD8⁺ CD45RC^|o T cells in peripheral blood. No significant difference was observed in splenic CD8⁺ CD45RC^|o T cells between DA, F344, and ACI rats (Figure 4D).

Cia5d regulates levels of the CD8⁺ CD45RC^|o CD25⁺ cells

In peripheral blood, most CD8⁺ CD45RC^|o and mature CD4⁺ CD45RC^|oT cells were CD25^+, and there were no significant differences between the parental strains (Figure 5A and Figure 5B). Although ACI rats had fewer splenic CD4⁺ CD45RC^|o T cells than DA rats (P = .038, Figure 4B), ACI rats expressed CD25⁺ more frequently than DA rats (P = .031, Figure 5C). The percentage of splenic CD8⁺ CD45RC^|oCD25⁺ cells was also higher in F344 and DA.F344(Cia5d) rats compared with DA rats (P = .066 and P = .005, respectively, Figure 5D). These results suggest that F344 rats are enriched for the CD8⁺ T regulatory cell subpopulation expressing both CD45RC^|o and CD25, and that Cia5d accounts for this difference.

Figure 5.

Frequency of CD25 cell surface expression within CD45RC^|o T cells. The frequency of CD4⁺ CD45RC^|o CD25⁺ and CD8⁺ CD45RC^|oCD25⁺ RTE and mature T cells in peripheral blood was similar in all strains (A–B). ACI rats had a higher percentage of CD25⁺ cells in splenic CD4⁺ CD45RC^|o mature T cells (C). The frequency of CD8⁺ CD45RC^|o CD25⁺ splenic cells was increased in F344 rats, and Cia5d accounted for this difference (D). Results are expressed as medians ± 25–75 percentiles for ACI (n = 10), DA (n = 10), F344 (n = 10), and DA.F344(Cia5d) (n = 9) rats. All results were compared with DA. **P ≤ 0.01; *P ≤ 0.05; †P = .066; Mann-Whitney U test.

ACI thymocytes are enriched for double-negative 2 (DN2) cells

We considered that thymocyte development could be genetically regulated, and differences in thymocytes could significantly affect peripheral blood and tissue numbers of T cells. Thymic cells from ACI, DA, and F344 rats were analyzed by flow cytometry. The frequency of thymocytes in the maturation stage DN2 was increased in ACI (P = .021), but not in F344 rats, compared with DA (Table 1) rats. The frequency of DN3 and DN4, double positive (DP), single positive (SP) CD4, and SP CD8 thymocytes was not significantly different between DA and F344 or ACI rats (Table 1). These observations suggest a unique intrathymic development homeostasis in ACI rats, with yet unclear peripheral consequences.

Table 1.

Frequencies of different thymocyte maturation stages, recent thymic emigrants, and in vitro cytokine production by stimulated T cells from DA, F344, and ACI rats^a

		DA	F344	ACI
Subset	Immunophenotype
DN2b	CD3-CD90+	39.6 (37.7–46.8)	33.4 (29.9–37.9)	55.9 (51.2–56.5)c
DN3 plus DN4d	CD3+ CD90+ CD4−CD8−	0.8 (0.2–1.1)	0.8 (0.2–0.4)	0.5 (0.4–0.8)
DPd	CD3+ CD90+ CD4+ CD8+	62.4 (40.7–81.7)	68.0 (47.0–83.3)	68.5 (40.7–79.2)
SP CD4d	CD3+ CD90+ CD4+ CD8−	4.3 (2.9–6.3)	3.3 (2.3–4.7)	5.4 (3.3–7.3)
SP CD8d	CD3+ CD90+ CD4−CD8+	29.0 (15.4–52.6)	28.9 (10.6–50.2)	25.6 (15.2–51.2)
RTE bloode	CD3+ CD90+	32.8 (30.6–39.7)	33.3 (28.2–44.2)	36.1 (28.8–45.6)
RTE spleene	CD3+ CD90+	37.3 (33.5–41.6)	37.2 (25.3–43.3)	33.9 (27.3–37.1)
Cytokinef	concentration
Interferon-γ	ng/mL	46.3 (39–65)	54.4 (47–62)	48.56 (48–48)
IL-10	pg/mL	344 (183–601)	671 (363–908)	232.5 (167–354)
TNFα	pg/mL	311 (207–353)	236 (164–324)	352 (296–366)

^aMedians (25–75 percentile).

^bPercentage of total thymocytes, DN = double negative.

^cP = .021, compared with DA (Mann-Whitney U test).

^dPercentage of CD3⁺ thymocytes cells; DP = double positive, SP = single positive.

^ePercentage of T cells; RTE = recent thymic emigrant.

^fMeasured in the supernatant of cultures stimulated with anti-CD3 and anti-CD28 antibodies for 72h.

Total numbers of circulating RTE in DA rats are similar to ACI and F344 rats

The overall frequency of RTE, defined as CD3⁺ CD90⁺ cells, did not differ between the strains tested (Table 1), and therefore we could not detect in rats the abnormalities in numbers of RTE previously reported in patients with RA.

DA, ACI, and F344 rats have similar T cells activation, proliferation, cytokine production, and AICD in response to in vitro stimulation

After 24 h of in vitro stimulation, the percentage of spleen and peripheral blood CD4⁺ CD25⁺ and CD8⁺ CD25⁺ T cells increased similarly in all three parental strains, reflecting similar activation thresholds (Figure 6). After 72 h of stimulation, T cell culture supernatants had similar levels of IFNγ and TNFα (Table 1). Levels of IL-10 were higher in F344 than in DA rats, but this difference did not reach statistical significance (Table 1). Levels of IL-4 were below the detection level (data not shown).

Figure 6.

In vitro T cell activation. Mature T cells were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 for 24h. CD4⁺ and CD8⁺ T cells from DA and F344 had similar frequency of CD25⁺ cells at baseline and 24h after stimulation in (A) peripheral blood and (B) spleen. Results are expressed as medians ± 25–sss75 percentiles. Results were compared with DA using the Mann-Whitney U test, and no significant differences were found.

All three parental strains had similar proliferative responses of CD4⁺ and CD8⁺ thymocytes, peripheral blood and splenic RTE, and mature T cells (Figure 7). AICD of peripheral blood, thymic, and splenic T cells increased similarly over time in all three strains (Figure 8). Taken together, these results show no significant differences in in vitro responses of stimulated naïve T cells, suggesting that intrinsic abnormalities in TCR-signaling in the presence of costimulation do not play a significant role in determining the arthritic phenotypes of these parental strains.

Figure 7.

In vitro T cell proliferation. T cells labeled with CFSE were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 for 72 h, and then analyzed by flow cytometry. The proliferative index is the average of divisions undergone by CFSE-labeled cells. Results are expressed as medians ± 25–75 percentiles. Results were compared with DA using the Mann-Whitney U test, and no significant differences were found.

Figure 8.

T cell susceptibility to activation-induced cell death. (A) Peripheral blood T cells, (B) spleen T cells, and (C) thymocytes were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 for 24, 72, or 96 h, then labeled with annexin-V and 7-AAD and quantified by flow cytometry. Double-negative cells were considered viable. Results are expressed as medians ± 25–75 percentiles. Results were compared with DA using the Mann-Whitney U test.

DISCUSSION

In the process of identifying arthritis severity and susceptibility genes, we hypothesized that at least some of these genes could regulate T cell phenotypes, including T cell development, homeostasis, and function. We included in our studies cell surface marker characteristics and in vitro responses previously described to be abnormal in peripheral blood of RA patients, such as numbers of RTE, CD4/CD8 numbers or ratios, and proliferative responses (35–37, 39–41). Additionally, several T cell characteristics including thymocyte maturation subsets, spleen and peripheral blood subpopulations, and T regulatory cells were analyzed because of their relevance to immune responses. A strong genetic influence on the regulation of several of these T cell characteristics was detected, where arthritis-susceptible and arthritis-resistant strains differed. We also observed for the first time that genetic loci (QTL) implicated in the regulation of arthritis severity also regulate numbers of CD4⁺ Treg (Cia5a) and CD8⁺ Treg (Cia5d) cells.

Arthritis-prone DA rats had increased CD4⁺ and decreased CD8⁺ T cell numbers compared with F344 rats, resulting in an increased CD4/CD8 ratio, similar to that reported in synovial and circulating lymphocytes of patients with RA (41). The fact that the CD4/CD8 ratio difference was also detected in RTE suggests that it derives from strain-specific variation in thymic output. Abnormalities in CD4/CD8 thymic lineage commitment are unlikely, because DA and F344 rats had similar frequencies of CD4⁺ and CD8⁺ single-positive thymocytes, suggesting differences in post-thymic homeostasis. Genes within the MHC have been shown to affect CD4/CD8 T cell ratio in the rat (42), but F344 and F344.DA(Cia1) rats (congenic for the DA MHC) had similar frequencies of CD4⁺ and CD8⁺ cells in peripheral blood and spleen, both of which were different from frequencies in DA rats, pointing to the effect of genes outside the MHC. Indeed, the frequencies of peripheral blood and splenic CD4⁺ and CD8⁺ T cells were accounted for by Cia5a. The overall lack of effect of Cia5a on numbers of CD4⁺ and CD8⁺ RTEs suggests that this gene regulates the number of mature T cells by modulating pathways related to post-thymic T cell homeostasis, such as peripheral expansion and/or cellular longevity. Intrinsic abnormalities in T cell proliferative responses and susceptibility to apoptosis were not supported by our in vitro studies. Hence, it is possible that the Cia5a effect on T cell turnover is indirect, for example operating via Treg cells, a hypothesis supported by data showing that Treg cells do regulate the numbers of CD4⁺ T cells (43) .

Results of several studies indicate that Treg cells are able to ameliorate autoimmune diseases in rodents (44), including arthritis (32,33,45,46). Rat natural Treg cells were originally described as CD45RC^|o TCR α/βCD4⁺ CD8⁻ CD90⁻ thoracic duct lymphocytes (47), and most of these cells were later recognized to be CD25⁺ (48,49), in agreement with Treg characteristics in mice (50) and humans (51). CD4⁺ CD45RC^|o CD25⁻ cells can also suppress the development of autoimmunity in vivo (48,49,52). These cells express PD-1, but not FoxP3 (52), and it remains unclear whether they are related to the CD4⁺ CD25⁻–inducible Treg cells (Th3 and Tr1) described in the mouse (53). We hypothesized that the number of Treg cells may be genetically regulated and thus would differ between DA and F344 or ACI rats, and that genetic control could be regulated by Cia5a or Cia5d.

The overall frequencies of CD25⁺ T cells in peripheral blood and spleen were similar in DA, F344, and ACI rats. However, the number of CD4⁺ CD45RC^|o cells in peripheral blood and spleen was increased in F344 rats compared with DA rats. In peripheral blood, but not in spleen, CD4⁺ CD45RC^|o cell numbers were regulated by Cia5a. We have previously shown that Cia5a regulates levels of pathogenic autoantibodies against type II collagen along with arthritis severity (14). Considering that CD4⁺ CD45RC^|o cells are capable of reducing levels of pathogenic antithyroglobulin antibodies along with the incidence of autoimmune thyroiditis in the rat (54), it is very possible that the arthritis and autoantibody regulatory effect of Cia5a is mediated via the regulation of CD4⁺ CD45RC^|o Treg cell numbers. Therefore, identifying the Cia5a gene may provide a novel way of increasing the generation of Treg cells to treat arthritis and perhaps other autoimmune diseases as well.

The number of CD4⁺ CD45RC^|o cells in arthritis-resistant ACI rats was consistently lower than in DA, and the significance of this finding remains to be determined. We also observed an increased number of CD4⁺ CD45RC^|o CD25⁺ cells in ACI, raising the possibility of a compensatory increase of this cell population, thus enabling adequate suppression of autoreactive cells and maintenance of the arthritis-resistant phenotype.

CD8⁺ Treg cells have been implicated in the induction of oral tolerance to various antigens (55), as well as in the regulation of oil-induced arthritis (56), experimental autoimmune encephalomyelitis (57), experimental autoimmune uveitis (58), allograft rejection (59), and graft-versus-host disease (60). CD8⁺ CD45RC^|o CD25⁺ Treg cells express FoxP3 and CTLA4 and suppress activity of CD4⁺ T cell responses by cell-to-cell contact (60–62). Our results revealed that F344 rats have increased numbers of splenic CD8⁺ CD45RC^|o CD25⁺ T cells compared with DA, and that this difference was accounted for by Cia5d. Therefore, Cia5d may control processes leading to the accumulation of CD8⁺ CD45RC^|o CD25⁺ cells in the spleen, such as preferential homing or increased local generation. Our data also revealed that numbers of peripheral blood CD8⁺ CD45RC^|o cells, which are a more heterogeneous population, were regulated by the MHC, in agreement with a previous study (63).

F344 rats had a markedly increased frequency of CD4⁺ CD25⁺ and CD8⁺ CD25⁺ thymocytes compared with DA rats. This difference in CD25⁺ thymocytes was accounted for by the MHC. These cells are thought to give rise to natural Treg cells and are capable of modulating immune responses in the rat (48,64,65) and in humans (66,67). MHC genes have a major role in the regulation of autoimmune arthritis in rodents (8) as well as in humans (68); nevertheless, nearly 30 years after the first report of the association of MHC with RA (69), it remains unclear how the MHC regulates disease susceptibility and severity. Our observations suggest a potentially novel model whereby arthritis-favoring alleles at the MHC could interfere with the thymic development of Treg cells.

Surface staining of thymocytes revealed that the DN2 compartment in ACI rats contained nearly 50% more cells than DA, whereas other thymic subpopulations were similar. Although the significance of this finding remains uncertain, DN2 cells are known to give origin to regulatory γ/δ T cells (70–72). Therefore, our observation raises the possibility that ACI DN2 thymocytes might have a reduced rate of apoptosis in vivo, or perhaps differentiate more frequently into regulatory γ/δ T cells. Because ACI and DA rats share the same MHC haplotype, this difference cannot be regulated by the MHC. Instead, numbers of DN2 cells may be regulated by non-MHC arthritis regulatory loci identified in DAxACI F2 studies (9,10,73).

Reduced numbers of RTE have been reported in RA, suggesting decreased thymic output (36,37). However, the similar percentage of CD90⁺ cells in peripheral blood and spleen indicates that baseline differences in thymic output are not genetically regulated in the DA, F344 and ACI parental strains and do not associate with the arthritic phenotype in these strains.

Increased T lymphocyte proliferative response (36,37) and resistance to AICD (74) were previously reported in RA. Additionally, mouse chromosome 11 loci syntenic to Cia5d have been implicated in the regulation of thymocyte proliferation (75), and T cell activation (76) and proliferation (77). While the relevance of these in vitro T cell responses for RA susceptibility and severity remains unclear, they were the basis for some of our experiments. We considered that, similarly to RA, arthritis-susceptible DA rats would have increased in vitro proliferative responses compared with arthritis-resistant ACI or F344 rats, and that Cia5d would account for differences between DA and F344. However, we observed no significant difference on T cell in vitro proliferative responses between strains. Levels of cytokines in the supernatant were also similar in all parental strains, although F344 T cells tended to produce more IL-10 than DA. CD4⁺ CD45RC^|o and CD8⁺ CD45RC^|o cells are known to release IL-10 upon stimulation (60,78, 9), and it is thus conceivable that the enrichment for these cells may have contributed to the moderately increased levels of IL-10.

The identification and characterization of the Cia5a and Cia5d arthritis severity genes will significantly increase our understanding of suppressive mechanisms operating in the pathogenesis of autoimmune arthritis. It will also provide strong candidate genes and pathways for case-control association studies in RA. Most importantly, the identification of these two genes will generate new biomarkers for prognostication, and novel targets for therapeutic interventions. Furthermore, Cia5a and Cia5d may identify a novel way of modulating Treg cells’ function or numbers, which will have direct relevance not only for arthritis and other autoimmune diseases, but also for cancer, transplantation and infectious diseases. In conclusion, we have identified for the first time arthritis QTL that regulate CD4⁺ CD45RC^|o (Cia5a) and CD8⁺ CD45RC^|o CD25⁺ (Cia5d) subsets of T regulatory cells. Additionally, we have determined that MHC genes significantly influence the frequency of the more heterogeneous CD8⁺ CD45RC^|o cell population in peripheral blood, and the frequency of CD25⁺ thymocytes, raising a novel potential explanation for the long-known MHC association with RA and other forms of autoimmune arthritis.