Perturbation of the T cell repertoire in rheumatoid arthritis

Abstract

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) beta-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR beta-chain sequences of 1 in 2.4 x 10(7) CD4 T cells. In RA patients, the median TCR beta-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Figure 1

Estimation of TCR β-chain frequencies. (A) Serial dilutions of a T cell clone with known TCR β-chain sequence were mixed with 1 × 10⁷ unrelated T cells. The mixtures were analyzed by nested BV- and BC/BJ-specific PCR followed by hybridization with the TCR N-D-N-specific probe. (B) TCR β-chain frequencies were determined in a limiting dilution system. A TCR β-chain sequence was chosen arbitrarily from peripheral blood CD4 T cells, and a TCR N-D-N specific probe was designed. Multiple serially diluted CD4 T cell samples were probed for the presence or absence of the particular TCR β-chain sequence by TCR BV-BC- and BV-BJ-specific PCR and subsequent hybridization. The frequency of the TCR β-chain was determined from the slope of the curve to be 1 in 8 × 10⁵ CD4 T cells.

Figure 2

Restricted TCR β-chain diversity in RA. (A) The frequencies of 36 arbitrarily chosen TCR BV8-BJ1S4 sequences from seven controls and 27 sequences from six RA patients were determined by limiting dilution analysis as shown in Fig. 1. Results are shown as box plots displaying medians and 25th and 75th percentiles. (B) TCR BV18-BJ2S5 sequences were analyzed from an additional five RA patients and five age-matched controls. Again, results are shown as box plots.

Figure 3

The influence of chronic stimulation with viral antigen on the diversity of the CD4 TCR repertoire. Frequencies of 17 BV8-BJ1S4 TCR β-chains from three patients with chronic hepatitis C infection were determined by limiting dilution analysis. Distributions of TCR β-chain frequencies in CD4 T cells are shown in comparison to normal donors and RA patients. The chronic antiviral response did not alter the diversity of the CD4 T cell repertoire.

Figure 4

Repertoire contraction in RA includes memory as well as naive CD4 T cells. TCR BV8-BJ1S4 sequences were obtained from purified CD4⁺ CD45RO⁺ memory (n = 22) and CD4⁺ CD45RO⁻ naive T cells (n = 36) from three RA patients and three controls, and the frequencies in the respective T cell subsets were determined. Compared with controls, TCR β-chain frequencies in RA patients were increased for the memory (P = 0.04) as well as the naive (P = 0.008) T cell subset.

Figure 5

Proliferative activity in CD4 T cells. Peripheral T cells were isolated freshly from normal controls and RA patients, and the percentages of CD4 T cells in the S-G2/M phase were determined. Results are shown as box plots. Median, 25th, and 75th percentiles (box), and 10th and 90th percentiles (whiskers) are displayed. The fraction of CD4 T cells in the cell cycle was increased significantly in RA patients (P = 0.01).

Figure 6

Telomeric shortening of CD4 T cells from RA patients. (A) A representative telomeric restriction blot from peripheral blood CD4 T cells of two RA patients (lanes 2 and 3, ages 42 and 43 years) and two controls (lanes 1 and 4, both age 40 years) is shown. (B) Summary of PhosphorImage analysis of terminal restriction fragment sizes from CD4 T cells of six RA patients and six age-matched normal controls ranging in age from 29 to 43 years.