Unchecked CD70 expression on T cells lowers threshold for T cell activation in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is characterized by premature immune aging with accumulation of degenerate T cells deficient for CD28. Gene expression profiling of CD4(+)CD28(-) and CD4(+)CD28(+) T cells to discover disease-promoting activities of CD28(-) T cells identified expression of CD70 as a most striking difference. Hence, CD70 was significantly more expressed in CD4 T cells from RA patients compared with age-matched controls (p < 0.006). The underlying mechanism was a failure to repress CD70 expression after activation-dependent induction. This defect in RA was not related to differential promoter demethylation. CD70 on bystander CD4(+)CD28(-) T cells functioned by lowering the threshold for T cell activation; admixture of CD4(+)CD28(-) T cells augmented TCR-induced responses of autologous naive CD4(+)CD28(+) T cells, particularly of low-avidity T cells. The data support a model in which CD70 expressed on T cells causes degeneracy in T cell responses and undermines tolerance mechanisms that normally control T cell autoreactivity.

Figure 1. Preferential expression of CD70 on CD4⁺CD28⁻ T cells

(A) Gene expression in CD4⁺CD28⁺ and CD4⁺CD28⁻ T cells was compared by Affymetrix Genechip Hu-95Av2. Amongst putative regulatory cell surface receptors, the largest difference was seen for the CD27–CD70 receptor-ligand pair. Data shown are the mean expression levels from three individuals. (B) Flow cytometric analysis of CD4⁺ T cell lines confirmed a CD70⁺CD27⁻ phenotype for CD4⁺CD28⁻ T cell lines. CD4⁺CD28⁻ T cells lacked activation markers such as CD69, suggesting that CD70 expression is not due to constitutive activation.

Figure 2. Kinetics of activation-induced CD70 expression on CD4⁺CD28⁺ and CD4⁺CD28⁻ T cells

PBMCs were stimulated with immobilized anti-CD3 and at the indicated time points stained with FITC-anti-CD70, PE-anti-CD28, PerCP-anti-CD4, and APC-anti-CD3 mAbs. Results are shown as histograms gated for CD4⁺CD28⁺ T cells (solid lines) and CD4⁺CD28⁻ T cells (gray shaded areas). Dotted lines represent stains with isotype control mAb.

Figure 3. DNA demethylation does not account for increased CD70 promoter activity

CpG methylation of the CD70 promoter of CD4⁺CD28⁺ and CD4⁺CD28⁻ T cells from RA patients before (A) and 4 days after (B) stimulation with immobilized anti-CD3 was assessed. CD28⁺ and CD28⁻ T cells showed similar methylation patterns; a representative example is shown. Closed circles (●) depict methylated CpG sites; open circles (○) represent unmethylated CpG sites. (C) Inhibition of DNMT activity (closed bars) with 1µM 5-Aza dC (left) or 10µM zebularine (middle) subsequent to anti-CD3 stimulation increased sustained CD70 expression, compared to untreated CD4 T cells (open bars). However, the relative difference between CD4⁺CD28⁺ and CD4⁻CD28⁻ T cells was maintained. Results are shown as mean ± SD of 6 experiments. Inhibition of the ERK pathway with 25µM PD98059 did not have an effect on CD70 expression (right).

Figure 4. Increased expression of CD70 on peripheral CD4 T cells from RA patients

CD70 expression on peripheral blood CD4 T cells was examined in healthy individuals (n=27) and RA patients (n=22) by multicolor flow cytometry. Data are shown separately for two age strata as box plots displaying medians, 25^th and 75^th percentiles as boxes, and 10^th and 90^th percentiles as whiskers.

Figure 5. Costimulatory function of CD4⁺CD28⁻ T cells

PBMCs were labeled with CFSE and stimulated with immobilized anti-CD3 in the presence of irradiated autologous CD4⁺CD28⁻ (A–C) or CD4⁺CD28⁺ T cells (D). Anti-CD70 mAb (B) or control Ig (C) was added to the CD4⁺CD28⁻ T cells before culture initiation. Cells were examined on day 5 of culture. Results are shown as histograms of gated CD4 T cells and are representative for an RA patient. Similar CD70-dependent costimulatory activity was seen with PBMCs of normal controls who had expanded CD4⁺CD28⁻ T-cell populations.

Figure 6. CD70-mediated costimulation preferentially targets naïve T cells

Purified naive (upper panel) and memory (lower panel) CD4 T cells were labeled with CFSE and stimulated with 1µg/ml immobilized anti-CD3 and 0.5µg/ml anti-CD28 in the presence or absence of irradiated CD4⁺CD28⁻ T cells. As indicated, CD4⁺CD28⁻ T cells were preincubated with anti-CD70 or isotype-matched control mAb for 1 hr before coculture. On day 5, cell divisions were assessed by CFSE dilution analysis. Data are representative of 3 experiments.

Figure 7. CD70 costimulation favors recruitment of low avidity CD4 T cells

(A) CFSE-labeled naïve CD4 T cells were stimulated with autologous DCs and increasing concentrations of TSST (0 to 100 ng/ml as indicated in the left-upper corner of each scatter plot). CFSE dilution was examined by flow cytometry for high (Vβ2⁺) and low (Vβ2⁻) avidity T cells. (B) CFSE-labeled naïve CD4⁺ T cells were cocultured with both autologous TSST-1-pulsed (1ng/ml) DCs and autologous CD4⁺CD28⁻ T cells (b and c) or CD4⁺CD28⁺ T cells (d). A negative control without TSST-1 is shown in (a). To confirm that the costimulatory effect of CD4⁺CD28⁻ T cells was related to their expression of CD70, cells were preincubated with anti-CD70 mAb (c) or with isotype control mAb (b). CFSE dilutions on Vβ2⁺ and Vβ2⁻ T cells were examined on day 5.