Cutting Edge: Antigen-Dependent Regulation of Telomerase Activity in Murine T Cells

Mice

Female C57BL/6 (B6) mice were obtained from Frederick Cancer Research Facility (Frederick, MD), and BALB/c mice were purchased from either Frederick Cancer Research Facility or The Jackson Laboratory (Bar Harbor, ME). Vβ8.1 TCR transgenic (Vβ8.1 Tg)³ mice (17) and BALB.Mls^a mice (18) were bred at Bioqual, Rockville, MD. DO11.10 TCR Tg mice (19) were bred at the University of Minnesota, Minneapolis, MI. All mice were used at 6 wk to 8 mo of age.

Preparation of T and B cell suspensions

Single cell suspensions were prepared from spleen and lymph nodes, as previously described (20). For in vitro cultures, splenic Vβ8.1 TCR Tg T cells were enriched by passage over rabbit anti-mouse-IgG-coated (Organon Teknika, West Chester, PA) tissue culture plates. APCs were prepared either from BALB/c.Mls^a (H-2^d, Mls^a-positive) or BALB/c (H-2^d, Mls^a-negative) mice that were injected with goat-anti-IgD serum (21), treated with anti-Thy-1.2 (HO-13-4 mAb, American Type Culture Collection (ATCC), Manassas, VA, TIB-99) and complement (C) (Pel-freez Biologics, Rogers, AK), and inactivated by mitomycin C treatment (Sigma, St. Louis, MO).

The adoptive transfer system using CD4⁺ DO11.10 Tg T cells has been described previously (22, 23). Briefly, spleen and lymph node cells from DO11.10 mice were treated with anti-CD8 mAb (83.12.5, ATCC) and rabbit C (Accurate Chemical and Scientific, Westbury, CT) and adoptively transferred into BALB/c mice. CD4⁺ DO11.10 Tg cells were isolated from the draining lymph nodes of recipient BALB/c mice by immunomagnetic selection using Dynabeads (Dynal, Oslo, Norway) precoated with rat anti-mouse IgG2a mAb and then with the Tg TCR-specific mAb KJ1.26 (24). The purity of the Tg⁺ cells was greater than 92%. Following exhaustive depletion of KJ1-26⁺ cells, recipient CD4⁺ Tg⁻ T cells were isolated by treatment with anti-CD8 mAb, rabbit C (Accurate Chemical and Scientific), and passage over a Cellect T cell column (Biotex Laboratories, Edmonton, Canada) to remove B cells. Greater than 85% of the Tg⁻ cells were CD4⁺.

In vitro stimulation cultures

The in vitro stimulation culture utilized in this report has been described previously (20).

In vivo stimulation of T cells

BALB/c mice were injected i.v. with 5 × 10⁶ CD8-depleted syngeneic T cells from DO11.10 TCR Tg mice and, within 24 h of cell transfer, immunized s.c. with 100 μg OVA (Sigma) in CFA (Sigma). As described above, CD4⁺ Tg⁺ and Tg⁻ T cells were isolated from pooled draining lymph nodes (LN) on days 0, 3, 5, and 15 after Ag immunization.

Reagents

Anti-B7-2 mAb (GL1 mAb, rat IgG2a) (25), anti-B7-1 mAb (1610.A1 mAb, hamster IgG,) (26), control rat IgG2a mAb (III/10 mAb) (20), and biotin-KJ1-26 were prepared by conventional methods. Goat-anti-IgD serum and anti-FcRγII (2.4G2 mAb) were the kind gifts of Dr. F. Finkelman (University of Cincinnati Medical Center, Cincinnati, OH) and J. Titus (National Institutes of Health, Bethesda, MD), respectively. ChromPure Hamster IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-anti-CD4, phycoerythrin-anti-CD4, biotin-anti-IL-2Rα, and biotin-anti-CD69 were purchased from PharMingen. SA-FITC and FITC-goat-anti-mouse IgG were purchased from Caltag Laboratories (Burlingame, CA). FITC-anti-Leu 4, biotin-anti-Leu 4, and biotin-anti-CD8 were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA) and Texas-Red-conjugated streptavidin (TRA) was purchased from Life Technologies (Gaithersburg, MD).

Telomeric repeat amplification protocol (TRAP)

The telomerase products were generated by a modification of the TRAP assay (10) described by Weng et al. (14). To quantitate telomerase activity, serial dilutions of telomerase extension products were assayed in the PCR reaction and used to determine linearity of the amplification reaction.

Cell cycle analysis

Cell cycle analysis of propidium iodide (PI)-stained (Sigma) cells was performed according to established methods (27).

In vitro Ag-specific and costimulation-dependent induction of telomerase activity

The regulation of telomerase activity in T cells has not previously been analyzed in responses to “physiologic” stimulation by Ag and costimulatory ligands. Therefore, to assess the ability of Ag-specific stimulation to induce telomerase in T cells, Mls^a-reactive Vβ8.1 TCR Tg T cells were stimulated with BALB.Mls^a APC and cultured for 1 to 3 days. Freshly isolated, resting T cells were found to have little or no detectable telomerase activity (data not shown). Vβ8.1 TCR Tg T cells stimulated with BALB.Mls^a (Mls^a-positive) APC Ag, in the presence of costimulation, were activated to express early activation Ags (CD69 and IL-2Rα), proliferated vigorously, and expressed high levels of telomerase activity (27-fold higher levels than that detected in unstimulated T cells) (Fig. 1⇓ and Table I⇓). Stimulation of Vβ8.1 TCR Tg T cells with BALB/c (Mls^a-negative) APC failed to elicit any of these activation-dependent events. We and others have previously shown that, in the presence of TCR stimulation, inhibition of APC-derived costimulatory signals by the addition of blocking B7-1- and B7-2-specific mAb substantially inhibited proliferation and IL-2 production without inhibiting the induction of CD69 and IL-2Rα expression (20). These results suggested that, in contrast to the induction of early activation Ags, proliferation and IL-2 production are dependent on B7 costimulatory signaling. In the present studies, these findings were confirmed. In addition, it was found that the induction of telomerase activity was strongly inhibited by the addition of a mixture of anti-B7-1 and anti-B7-2 mAbs, indicating that this response, like proliferation and IL-2 production, is costimulus dependent. Consistent with this interpretation, the proliferative responses and telomerase activity of Mls^a-stimulated Tg T cells deprived of costimulatory signals were both sustantially restored by the addition of exogenous IL-2 (Table I⇓ and Fig. 1⇓). Similar results were obtained when T cells were stimulated with soluble anti-CD3ε mAb under conditions requiring APC-dependent costimulation (data not shown). The requirement for costimulatory CD28-B7 interactions in the induction of telomerase activity was also supported by the observation that T cells from CD28-deficient mice failed to express telomerase in response to APC-dependent TCR signaling (data not shown). Taken together these results demonstrate that optimal induction of telomerase activity and proliferative responses by T cells appear to require both TCR signals and costimulatory signals that can be provided by CD28-B7 interactions.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Comparison of telomerase activity induced in Mls^a-specific Vβ8.1 TCR Tg T cells stimulated in vitro with Mls^a-expressing BALB.Mls^a APC in the presence of control or B7-1- and B7-2-specific mAb. A, Cells were cultured for 1, 2, or 3 days, and 2 × 10³ cell equivalents were analyzed for telomerase activity. B, Relative telomerase activity detected by serial threefold dilutions of telomerase extracts prepared from 2 × 10³ cells harvested after 3 days of culture.

In vivo induction of telomerase activity

Regulation of telomerase activity in T cells in response to defined antigenic challenge in vivo has not previously been evaluated. This issue was therefore addressed in an adoptive transfer system employing CD4⁺ T cells from DO11.10 TCR Tg mice expressing a TCR specific for OVA peptide + IA^d (22, 23). CD4⁺ Tg⁺ and Tg⁻ T cells were isolated from draining lymph nodes of adoptively transferred mice 0, 3, 5, and 15 days after OVA + CFA immunization. The number of CD4⁺ Tg⁺ T cells recovered 3 to 5 days after immunization was increased five- to ninefold over starting numbers, and a high proportion of the Tg⁺ cells recovered on day 3 were in S, G2, and M phases of the cell cycle (Table II⇓), indicating that substantial clonal expansion of Tg⁺ cells was induced by in vivo Ag challenge. This result is consistent with previous reports demonstrating that Ag induced adoptively transferred DO11.10 cells to proliferate and to express cell surface activation markers through a pathway that is dependent on CD28-B7 interactions (23, 28). As shown in Figure 2⇓, Tg⁺ CD4⁺ T cells isolated at day 0 expressed low or undetectable levels of telomerase activity. In contrast, adoptively transferred Tg⁺ CD4⁺ T cells isolated 3 or 5 days after Ag stimulation expressed levels of telomerase activity at least 18- to 36-fold higher than that observed in unimmunized cells. By day 15, telomerase activity in these Tg⁺ T cells had diminished. In contrast to the substantial induction of telomerase activity in Tg⁺ T cells, Tg⁻ CD4⁺ T cells isolated from these same mice had no significant increase in telomerase activity over the low level seen at day 0 (Fig. 2⇓B). Thus, the in vivo expression of telomerase activity in T cells was dependent both upon expression of Tg TCR and Ag activation. In addition, these results demonstrate that in vivo Ag-specific stimulation of CD4⁺ Tg⁺ T cells concomitantly induces high levels of telomerase activity as well as extensive cell division and clonal expansion.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Comparison of telomerase activity induced in vivo in Tg⁺ and Tg⁻ CD4⁺ T cells. A, Telomerase activity in in vivo Ag-stimulated Tg⁺ CD4⁺ T cells. Adoptively transferred CD4⁺ Tg⁺ T cells were isolated from mice that had been immunized with OVA + CFA 0, 3, 5, or 15 days previously. Telomerase activity was detected by serial dilutions of telomerase extracts. The 1:1 dilution shows telomerase activity present in 2 × 10³ cell equivalents. B, Relative telomerase activity detected by serial threefold dilutions of telomerase extracts prepared from 2 × 10³ Tg⁺ and Tg⁻ CD4⁺ T cells isolated 0, 3, 5, and 15 days after Ag immunization.