Telomere length of in vivo expanded CD4⁺CD25⁺ regulatory T-cells is preserved in cancer patients

Abstract

Purpose: CD4⁺CD25⁺ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the upregulation of telomerase activity, whose regulation also remains unknown for Treg. Experimental Design: Treg and CD4⁺CD25⁻ T-cells were isolated from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17) and analyzed for their content of T-cell receptor excision circles (TREC) and for telomere length using flow-FISH, real-time PCR and Southern blotting. The in vitro regulation of telomerase of Treg was studied using PCR-ELISA in bulk cultures as well as in isolated proliferating and non-proliferating Treg. Results: Treg isolated from peripheral blood of cancer patients exhibit significantly decreased levels of TREC when compared to Treg from healthy controls. Despite their in vivo proliferation, telomere length is not further shortened in Treg from cancer patients. Accordingly, telomerase activity of Treg was readily inducible in vitro. Notably, sorting of in vitro proliferating Treg revealed a significant telomere shortening in Treg with high-proliferative capacity. The latter are characterized by shortened telomeres despite high telomerase activity. Conclusions: Increased frequencies of Treg in peripheral blood of cancer patients are due to active proliferation rather than due to redistribution from other compartments (i.e., secondary lymphoid organs or bone marrow). In vivo expansion does not further shorten telomere length, probably due to induction of telomerase activity. In contrast, under conditions of strong in vitro stimulation telomerase induction seems to be insufficient to avoid progressive telomere shortening.

Introduction

There is compelling evidence that T-cells co-expressing CD4 together with the IL2-receptor alpha chain (CD25), the so-called CD4⁺CD25⁺ regulatory T-cells (Treg), are key players for the maintenance of tolerance in rodents and humans [23]. In addition, Treg might also be involved in the dysregulation of the immune response under several pathological conditions. Accordingly, patients suffering from multiple sclerosis (MS) [29] and autoimmune-mediated mixed cryoglobulinemia in hepatitis C-positive individuals [5] display a numerical and functional Treg deficit. These examples demonstrate that under conditions of a decreased immune tolerance to autoantigens, a decrease of Treg number and/or function might promote the development of autoimmune diseases.

In contrast, patients suffering from cancer, who require a functional T-cell-mediated anti-tumor immune response, have a significantly enlarged Treg pool in the peripheral blood, in tumor-infiltrating lymphocytes (TIL) as well as in tumor-draining lymph nodes [24, 26, 33, 34]. With respect to the findings that Treg-depleted mice have an enhanced anti-tumor immunity [18, 25], Treg expansion might depict an important immune-evasion mechanism in cancer patients. It is noteworthy that, at least in animal models, a tumor antigen-specific Treg expansion has recently been shown [30]. Yet, the mechanisms causally involved in the observed expansion of Treg in cancer patients remain to be determined.

The expandability of isolated Treg in vitro has been convincingly reported by a variety of research groups [10, 33]. The requirements for successful in vitro expansion of Treg comprise the supplementation of IL-2 in addition to T-cell receptor stimulation, as IL-2 has been shown to abrogate the anergic state of Treg [9]. The in vitro expandability of Treg appears of great relevance for future therapeutic approaches dealing with the application of autologous in vitro expanded Treg for the treatment of autoimmune disorders and GvHD after allogeneic bone marrow transplantation.

Telomeres are genetic elements that are essential for the stability of the chromosomal ends. Their critical shortening or complete loss leads to the formation of unstable end-to-end fusions resulting in chromosomal instability and growth arrest [4, 8]. Telomeric chromatin is composed of a multitude of telomeric protein-binding sites and tandem arrays of simple DNA repeats (TTAGGG)_n in mammals [15]. The telomerase complex is required for the complete replication of the telomeric ends of the chromosome during each cell division. It is a characteristic feature of immortal cells, such as tumor or germline cells, that they constitutively express the telomere-extending enzyme telomerase, whereas telomerase activity is absent in normal somatic cells [3, 8, 12, 16]. However, recent studies have shown that B- and T-cells are an exception to that rule. During activation, both T- and B-cells are capable of upregulating telomerase in a tightly regulated fashion [31].

The lifespan and replicative potential of T-cells has been shown to be determined by telomerase function. This has been demonstrated by overexpression of hTert [22] as well as by blocking experiments in T-cell lines using a dominant negative (DN) hTert vector construct [20]. The latter decreased the life span of T-cells in culture, whereas the overexpression of hTert enabled immortalization of CD8⁺ T-cells. Telomerase function also appears to be critical for the prevention of genomic aberrations in T-cells thereby ensuring genomic stability under conditions of expansion. A recent report also suggests a critical role of telomerase activity in regulatory T-cell lines [14]. To date, however, there is no data available on the regulation of hTert expression and telomerase function in primary human Treg.

Our current report provides first time evidence that the increase of Treg in peripheral blood of cancer patients is indeed due to cell proliferation, as they contain lower levels of TREC when compared to Treg isolated from healthy controls. Under conditions of in vivo Treg expansion in those patients, telomere length of Treg is preserved when compared to age-matched healthy control subjects. Accordingly, telomerase function can be rapidly upregulated in Treg isolated from peripheral blood under conditions of in vitro expansion. However, telomerase activity is induced only in the proliferating Treg pool, whereas non-proliferating Treg exhibited low telomerase activity. This finding indicates a central role of telomerase induction for Treg expansion, albeit under conditions of extensive in vitro stimulation telomerase induction is obviously not sufficient to maintain the telomere length in the proliferating cell fraction.

Material and methods

Isolation of peripheral blood CD4⁺CD25⁺ and CD4⁺CD25⁻ T-cells

Heparinized venous blood was collected from 17 healthy donors (58.2±8.2 years) and 23 patients suffering from epithelial malignancies (63.9±9.9 years) after informed consent was obtained. Further patient characteristics are listed in Table 1. All cancer patients were newly diagnosed and neither received immuno-suppressive drugs nor chemotherapy. CD4⁺CD25⁺ and CD4⁺CD25⁻ were magnetically separated from PBMC using the MACS Treg kit (Miltenyi, Bergisch Gladbach, Germany). Using optimized amounts of the anti-CD25 mAb, purity routinely achieved > 90% as confirmed by FACS.

Table 1.

Patients’ characteristics

Caner type	Stage 3–4 (n)
Colon	3
Gallbladder	1
Gastric	1
Breast	3
Lung	13
Colorectal	2
Total	23

Flow cytometry (FACS)

All antibodies (mAbs) used for FACS analysis were purchased from Becton Dickinson (San Diego, CA, USA). The following antibodies were used in this study: anti-CD4 APC, anti-CD25 PE, CD69 FITC and anti-CD45RO CyChrome. Prepared cells were incubated with the appropriate fluorochrome-linked mAb or the corresponding isotype control for 15 min at room temperature. After washing, data were acquired using a FACSCalibur flow cytometer (Becton Dickinson) and then analyzed using CELLQUEST software.

Membrane labeling of T-cells with carboxy fluorescein succinimidyl ester (CFSE)

T-cells were resuspended in PBS at 2×10⁶/ml. CFSE (Sigma Chemicals, St. Louis, MO, USA) was added at the final concentration of 0.5 mmol/L for 15 min at 37°C. Finally, T-cells were washed twice in PBS/2% BSA and cultured in RPMI/10% FCS.

T-cell culture

The 2×10⁶ CFSE-labeled Treg/ml were cultured with 2 μg/ml plate-bound anti-CD3 (OKT3) together with 50 U/ml IL-2 for 2–14 days. Stimulation with anti-CD3 plus IL-2 results in cell division with distinct CFSE fluorescence peaks, which allows the discrimination between cycled and non-cycled cells by FACS analysis.

Cell sorting

Sorting of proliferating (CFSE^low) versus non-proliferating (CFSE^high) CD4⁺CD25⁺ and CD4⁺CD25⁻ cells from anti-CD3 plus IL-2-stimulated T-cell cultures was performed at days 4–7 by using a FACSVantage (Becton Dickinson). Sorted fractions were either subjected to DNA or protein purification or were used in suppression assays as described below.

Suppression assays

To determine the regulatory properties of the sorted cell fractions, co-cultures of 1×10⁵ sorted and then irradiated CFSE^high or CFSE^low Treg or simultaneously expanded CD4⁺CD25⁻ together with 1×10⁵ allogeneic CD4⁺CD25⁻ responder cells were performed in OKT-3 pre-coated wells (2 μg/ml). As control the respective sorted populations or the responder cells were cultured alone. Proliferation was measured at day 5 by ³H-thymidine incorporation in a ß-scintillator.

Trap assay

Telomerase activity was monitored using the telomerase PCR ELISA kit (Roche Molecular Biochemicals, Vienna, Austria) strictly according to the manufacturer’s instructions. In brief, 2×10⁵ cells were pelleted and 200 μl of lysis reagent was added. After 30 min at 4°C, lysates were centrifuged and equal amount of protein was used for each telomeric repeat PCR amplification reaction. The amplification product was visualized using a colorimetric method. Absorbance values are reported as absorbance at 450 nm read against the blank value (reference wavelength 620 nm).

Determination of telomere length by real-time PCR

DNA was extracted by the use of the QiaAmp Blood Mini Kit (Qiagen, Vienna, Austria). Prior to real-time PCR procedure samples were measured for DNA content using a commercial available DNA quantification Kit (PicoGreen® dsDNA Quantitation Kit, Molecular Probes; USA). Samples were aligned to the same quantity and transferred to an optical reaction plate (PE Biosystems). A 12-fold standard was added to each plate. A reaction mix containing Tris–HCl, KCl, DMSO (Sigma Chemicals), SYBR-Green (Eubio, Vienna, Austria), dNTPs (Amersham Biosciences, Piscataway, NY, USA), MgCl₂, AmpliTaq Gold DNA Polymerase (Applied Biosystems) and two primer pairs (MWG-Biotech AG, Germany) were added to each sample at 4°C. The telomere-specific primer pair is a modification of previously published primers [7]. The reference primer pair 36B4 is a single copy gene encoding for acidic ribosomal phosphoprotein PO, located on chromosome 12. The cycle threshold of samples and standard was determined using SDS 1.9.1. As an internal control a melting curve was added to each run to control primer dimer formation.

Telomere length is determined by generation of a mean telomere/reference gene ratio for each sample. The relative length of the telomere can be measured by comparing the differences in the cycle threshold number between reference gene and telomere allowing a semi-quantitative analysis of the telomere length.

Determination of telomere length by Southern blotting

After quantification of genomic DNA, 1.5 μg DNA was digested with frequently cutting enzymes (Hinf I, Rsa I). DNA fragments are separated on a 0.8% agarose gel (Agarose MP; Roche Diagnostics, Germany). After DNA-blotting onto a nylon membrane, telomeric repeat sequences were hybridized using complementary digoxigenin (DIG)-labeled oligonucleotides. By metabolizing a highly sensitive chemiluminescent substrate, DIG-labaled telomere repeat sequences can be visualized with a Lumi-Imager. By using a DNA length standard the average size of the telomeric repeat fragments was determined. For our experiments a commercially available Kit (TeloTAGGG Telomere Length Assay, Roche Diagnostics; Germany) was used. For Southern Blot experiments 1.5 μg DNA of each sample was inserted. Telomere length was analyzed by a Flour-S (TM)-Multiimager (Bio Rad; Hercules, CA, USA) using the Quantity One 4.1.1 software.

Determination of telomere length by flow-FISH

Telomere length was determined using the flow-FISH technique as described recently [19]. In brief, cells were washed twice in PBS followed by a wash in 1 ml hybridization buffer [70% deionized formamide, 20 mM Tris pH 7.1% BSA (all from Sigma Chemicals)]. Samples were resuspended in 200 μl hybridization buffer and then incubated with 0.3 μg/mL of the Cy5-conjugated PNA telomeric Probe (C₃TA₂)₃ (Applied Biosystems) for 10 min followed by heating at 82°C for 10 min. Cells were cooled on ice and further hybridized for 2 h at room temperature. Samples were washed twice in post-hybridization buffer (70% formamide, 10 mM Tris, 0.1% BSA, 0.1% Tween 20 [all from Sigma Chemicals]) and PBS. Samples were then subjected to flow cytometry and finally analyzed using Cell Quest Software. Polyfluorescent beads were used at the beginning of each experiment to standardize the cytometer. Following the final wash, samples were incubated with propidium iodide (5 μg/mL; Sigma Chemicals) and RNAase (50 ng/mL; Sigma Chemicals) for 15 min to exclude cell doublets.

Determination of hTert and FoxP3 mRNA by real-time PCR

Real-time PCR was run on a TaqMan System (PE Biosystems). Primers for hTert were as follows: sense 5′-TGGCTGGGAAAATGGCA-3′; reverse 5′-GCAGGAGCCCTTGTCGG-3′; hTert TaqMan Probe 5′-fluorescent dye 6-carboxyfluorescein (FAM)-TGACCAAGGCTTCATCTGTGGCATCA-6-carboxy-tetramethyl-rhodamine (TAMRA)-3′. Primers for FoxP3 were as follows: 5′-TGG CTA GGA AAA TGG CA-3′; reverse 5′-GCA GGA GCC CTT GTC GG -3′; Probe 5′ FAM-TGA CCA AGG CTT CAT CTG TGG CAT CA-TAMRA-3. For quantification of the human housekeeping gene GAPDH, the Perkin Elmer pre-developed assay kit was used. The amplification was carried out and analyzed in the AbiPrism 7700 Sequence detector. Real-time PCR efficiencies were acquired by amplification of a standardized dilution series of cDNA from K562 cells. The amount of hTert and FoxP3 was calculated by the method of Pfaffl.

Quantification of T-cell receptor excision circles (TREC) by real-time PCR

The number of TREC was determined by quantitative real-time PCR using the ABI PRISM 7700 Sequence Detector (PE Biosystems). In brief, the percentage of cells carrying TREC was determined by using a duplex vector containing a fragment of the δRec-ϕJα signal joint (TREC) and a fragment of the RAG2 gene (kindly provided by C.A. Schmidt, University of Greifswald) as a reference. Based on the DNA concentration, standard dilutions of the vector from 10⁷ to 10¹ copies were prepared. In brief, PCR of 50 μl total volume was performed with ~100 ng of genomic DNA, 25 pmol of each primers, 10 nmol each dNTP, 1.25 U Platinium Taq polymerase, 5 pmol of 6FAM-TAMRA probe and PCR buffer including 4.5 mM MgCl₂. After the initial denaturation at 95°C for 5′, 45 cycles consisting of 95°C for 30 s and 66°C for 30 s were performed. For TREC analysis the 5′ primer ϕJα (−258): AAC AGC CTT TGG GAC ACT ATC G and the 3′ primer δRecsj(+104): AAC AGC CTT TGG GAC ACT ATC G, amplifying the signal joint sequence generated by the δRec-ϕJα rearrangement, were used together with the TREC probe: 6FAM-CCA CAT CCC TTT CAA CCA TGC TGA TGA CAC CTC T-TAMRA. For RAG2 analysis the 5′ primer: RAG2(2160) GCA ACA TGG GAA ATG GAA CTG, the 3′ primer: RAG2(2404) GGT GTC AAA TTC ATC ATC ACC ATC and the RAG2 probe: 6FAM-CCC CTG GAT CTT CTG TTG ATG TTT GAC TGT TTG TGA-TAMRA were used. The content of TREC was calculated as TREC content per μg DNA.

Statistical analysis

After the analysis of variance, Student’s t test was used. P values <0.05 were considered significant. Statistical analysis was performed using GraphPadPrism software.

Results

Treg have shorter telomeres than CD4⁺CD25⁻ T-cells

To analyze the telomere length of Treg, CD4⁺CD25⁺ T-cells were isolated from the peripheral blood of healthy volunteers using magnetically labeled beads. Figure 1 demonstrates that Treg have significantly shorter telomeres when compared to the CD4⁺CD25⁻ T-cell population, which served as the internal reference. The data obtained by quantitative RT-PCR on genomic DNA (Fig. 1a) were corroborated by Southern blotting (Fig. 1b) as well as by flow-FISH (mean fluorescent intensity of CD4⁺CD25⁺ T-cells was reduced by 14.5±5.1% vs. CD4⁺CD25⁻ T-cells, P<0.05; see further Fig. 2c).

Fig. 1.

Telomeres of Treg are significantly shorter when compared to CD4⁺CD25⁻ T-cells. Treg and the respective control population, i.e., CD4⁺CD25⁻ T-cells were isolated from peripheral blood of healthy volunteers by magnetic sorting. Telomere length was analyzed by a panel of different methods. a Genomic DNA was isolated and used as template for quantitative real-time PCR for telomere length measurement (*P≤0.05). b Absolute telomere length as determined by Southern blotting (n=4) and scanning densitometry. Representative Southern blotting of the respective cell fractions of two individuals are shown. The size marker is given on the right lane

Fig. 2.

Treg from cancer patients are increased in the peripheral blood and express FoxP3. a A representative staining of Treg in a healthy control and a cancer patient is shown. CD25 and CD45RO staining of CD4⁺ and CD69⁻ gated T-cells is given. b FoxP3 was analyzed by real-time PCR after isolation of CD4⁺CD25⁻ T-cells and Treg from peripheral blood of cancer patients and the respective healthy controls

Telomere length is preserved under conditions of in vivo expansion

To gain deeper insight in the regulation of telomere length in Treg, telomere length of Treg isolated from patients with newly diagnosed locally advanced or metastatic epithelial malignancies (which did not receive any chemotherapeutic or immuno-suppressive therapy) were analyzed in comparison with healthy age-matched control subjects. The enlargement of the Treg pool was proven in each patient by staining of CD4, CD25, CD45R0 as well as CD69 (Fig. 2a) in the peripheral blood as described previously [33]. In addition, each isolated CD4⁺CD25⁻ and CD4⁺CD25⁺ T-cell fraction was subjected to quantification of the Treg-specific transcription factor FoxP3 by means of real-time PCR. CD4⁺CD25⁺ T-cells expressed significantly higher levels of FoxP3, further corroborating the regulatory nature of the cells (Fig. 2b). However, when we determined the telomere length of isolated Treg from cancer patients by flow-FISH, we were not able to detect a significant shortening of telomere length (Fig. 3a) as compared to Treg isolated from age-matched healthy individuals when compared to the respective control cells, i.e., CD4⁺CD25⁻ T-cells. The mean telomere length in healthy subjects and cancer patients determined by analysis of the mean fluorescence intensity is given in Fig. 3b. The data were corroborated by real-time RT-PCR (Fig. 3c) and did not show any statistically difference when compared to telomere length difference between Treg and control cells from healthy control subjects (see Fig. 1a).

Fig. 3.

Telomere length in Treg isolated from cancer patients is preserved. a Telomere length was analyzed by flow-FISH after isolation of Treg and CD4⁺CD25⁻ T-cells from cancer patients and age-matched controls. Flow-FISH data from a representative matched-pair are shown. b The MFI of the internal control cell population (i.e., CD4⁺CD25⁻ T-cells) obtained by flow-FISH was set as 100%. The mean reduction of telomere length of cancer patients and the respective controls is given (*P≤0.05). c Genomic DNA was isolated from Treg and CD4⁺CD25⁻ T-cells obtained from epithelial cancer patients and telomere length was subsequently determined by real-time PCR. The mean reduction of telomere length of Treg in cancer patients as compared to the respective control CD4⁺CD25⁻ T-cell population is shown (*P≤0.05)

Treg from cancer patients have a lower TREC content when compared to age-matched healthy controls

We and others demonstrated an increased Treg content in the peripheral blood of cancer patients [33, 34]. However, it is still unclear whether this observation is due to active cycling of Treg or whether it rather reflects redistribution of Treg from other compartments such as secondary lymphoid organs or bone marrow. To address this critical issue, we applied real-time PCR for quantification of TREC in isolated Treg from untreated cancer patients and healthy control subjects. Purified Treg from cancer patients are characterized by a significantly lower content of TREC as compared to Treg isolated from age-matched healthy volunteers (Fig. 4), pointing to the fact that these cells indeed actively proliferated in vivo. Only a slight but not significant decrease was observed between the CD4⁺CD25⁻ T-cell fractions.

Fig. 4.

Treg from cancer patients have a lower TREC content when compared to age-matched healthy controls. DNA was isolated from Treg and from CD4⁺CD25⁻ T-cells of either epithelial cancer patients or healthy age-matched controls. TREC content of the respective cell population is given as TREC copies per μg DNA (*P≤0.05 for CD4⁺CD25⁺ from healthy controls vs. CD4⁺CD25⁺ from cancer patients)

Telomerase activity is rapidly induced in Treg under conditions of in vitro expansion

It is well known that Treg can be expanded in vitro by T-cell receptor stimulation in combination with IL-2. We therefore studied the regulation of telomerase activity under these culture conditions. Figure 5a demonstrates that Treg are characterized by a significant induction of telomerase activity as early as 4 days after the start of the culture (which correlated with appearance of proliferating T-cell clones in the culture). The maximum of telomerase activity was lower when compared to cultured CD4⁺CD25⁻ T-cells, which correlated with the lower proliferative potential of the CD4⁺CD25⁺ T-cell fraction proven by ³H-Thy incorporation (data not shown) and CFSE-staining (see below).

Fig. 5.

Telomerase activity and hTert mRNA expression in expanding CD4⁺CD25⁺ T-cells and CD4⁺CD25⁻ T-cells. Freshly isolated CD4⁺CD25⁺ T-cells and CD4⁺CD25⁻ obtained from healthy controls were in vitro expanded by plate-bound OKT-3 in combination 50 U/ml IL-2. At the indicated time points, protein extract was prepared. a The mean value of four independent experiments showing telomerase activity for both cell populations as determined by PCR-ELISA is shown. The induction was normalized to telomerase activity determined after fresh isolation, which was arbitrarily set to 1. b Total RNA was isolated from CD4⁺CD25⁺ T-cells and CD4⁺CD25⁻ T-cells. hTert mRNA was analyzed by real-time PCR (*P≤0.05, n=8). hTERT expression is given as x-fold versus hTERT expression of the K562 cells, which was arbitrarily set to 1

Treg are characterized by a lower hTert mRNA expression as compared to CD4⁺CD25⁻ T-cells

mRNA was obtained from freshly isolated Treg as well as from the respective CD4⁺CD25⁻ control cell population. Quantitative RT-PCR revealed that Treg have a significant lower expression of hTert mRNA as compared to CD4⁺CD25⁻ cells (Fig. 5b). The erythroleukemic cell line K562, which is known to express high levels of hTert mRNA and telomerase activity, was used as positive control.

Telomerase activity is primarily induced in the proliferating pool of CD4⁺CD25⁺ T-cells

Figure 6a depicts the expandability of Treg in vitro. However, it is not clear whether the proliferating part is indeed immuno-suppressive, as there remains even under anti-CD3 and IL-2 stimulation a non-proliferating, anergic cell population (see gate R1 of right histogram in Fig. 6a). It is therefore conceivable that the non-proliferating fraction of a Treg bulk culture exerts the immuno-suppression, whereas the proliferating part does not. We therefore labeled isolated Treg with CFSE and subsequently expanded the cells for 7 days. After FACS-sorting, two cell populations designated CFSE^high (non-proliferating) and CFSE^low (proliferating) were obtained (the sorting strategy is demonstrated in Fig. 6b). To ensure the immuno-suppressive activity of the expanded cell population (CFSE^low), the isolated cell fractions were irradiated and co-cultured with CD4⁺CD25⁻ T-cells. Figure 6c corroborates the immuno-suppressive properties of the proliferating proportion of in vitro expanded Treg.

Fig. 6.

Sorting strategy for the isolation of proliferating and non-proliferating CD4⁺CD25⁺ T-cells. Freshly isolated CD4⁺CD25⁺ and CD4⁺CD25⁻ T-cells obtained from healthy controls were labeled with CFSE followed by in vitro stimulation with plate-bound OKT-3 in combination with 50 U/ml IL-2. A characteristic CFSE staining profile of both cell populations is depicted in a. On day 7 two cell populations were sorted: CFSE^high (b, right gate) and CFSE^low (b, left gate). Purity was controlled by FACS. The immuno-suppressive property of the sorted cell fractions was corroborated by co-cultures of CD4⁺CD25⁻ T-cells (responder cells) together with 1×10⁵ sorted and then irradiated CFSE^high or CFSE^low T-cells (suppressor cells) or CD4⁺CD25⁻ T-cells obtained from healthy controls. Proliferation was measured at day 5 by ³H-thymidine incorporation (*P≤0.05) (c)

We then analyzed telomerase activity in CFSE^high and CFSE^low cells. A significant induction of telomerase activity was detectable in the CFSE^low fraction. In contrast, although detectable, the induction of telomerase activity was very weak in the CFSE^high cells (Fig. 7a). The observation that CFSE^low Treg have a higher telomerase activity, whereas the unsorted fraction is characterized by a lower telomerase function is most likely due to the lower percentage of CFSE^low Treg as compared to CFSE^low CD4⁺CD25⁻ T-cells 7 days after in vitro expansion of bulk cultures (see Fig. 5a).

Fig. 7.

Telomerase activity in primarily induced in CFSE^low Treg but is not sufficient to maintain telomere length in vitro. a Protein extracts of freshly isolated Treg, CD4⁺CD25⁻ T-cells and the corresponding expanded CFSE^high and CFSE^low populations were tested for telomerase activity by PCR-ELISA. One representative experiment out of three is shown. The induction of telomerase activity is normalized to telomerase activity on day 0 (after isolation) which is arbitrarily set to 1 for both cell populations. The shortening of telomeres within the CFSE^low fraction as compared to the CFSE^high fraction of in vitro expanded CD4⁺CD25⁺ T-cells is shown by (b) flow-FISH and (c) real-time PCR. Representative results of the CFSE^low and the CFSE^high fraction from three independent experiments are shown

Telomerase induction does not prevent telomere shortening in the CFSE^low fraction in vitro

We subsequently analyzed the telomere shortening of proliferating Treg under conditions of in vitro expansion. As shown in Fig. 7b, the CFSE^low fraction Treg is characterized by a significant telomere shortening under conditions of extensive in vitro expansion. The data were corroborated by real-time PCR (exemplarily shown for the CFSE^high and the CFSE^low fraction of expanded Treg in Fig. 7c). This observation indicates, at least under the given in vitro conditions and the limited time-span of our experiments, that there is a subpopulation of proliferating Treg characterized by a significant telomere shortening despite rapid induction of telomerase activity.

Discussion

In this report we demonstrate that telomere length of CD4⁺CD25⁺ regulatory T-cells (Treg) isolated from cancer patients is preserved, despite their significant loss of TRECs, which provides for the first time evidence that the observed increase of Treg in cancer patients is indeed due to active proliferation rather than due to redistribution of Treg from secondary lymphoid organs or bone marrow. Data from in vitro experiments further demonstrate that telomerase activity can be rapidly induced in Treg upon T-cell receptor stimulation in combination with IL-2. Of note, telomerase activity is primarily induced in the proliferating pool of in vitro expanded Treg which suggest a central role of telomerase induction for a robust in vitro proliferation. However, in contrast to the in vivo situation in cancer patients, telomerase induction is not sufficient for maintenance of telomeres in the proliferating pool of Treg under conditions of strong in vitro stimulation.

Recently, Taams and co-workers [27] hypothesized that Treg are, at least in part, differentiated from CD4⁺CD45RO⁺RB^high memory T-cells by repeated antigen encounter. The author’s concept is supported by in vitro data showing that continous stimulation of CD4⁺CD25⁻ T-cells with a given antigen in combination with TCR-stimulation enables the generation of regulatory T-cells. This process is paralleled by a significant shortening of telomeres in the generated regulatory T-cells. Thus, these cells can be considered as highly differentiated and/or senescent T-cells. This assumption is supported by a very recent paper describing that Treg isolated from peripheral blood exhibit a significant loss of TREC, demonstrating that these cells indeed underwent multiple rounds of post-thymic cell divisions [11]. On the other hand, Treg are known to be increased in vivo in cancer patients [24, 26, 33, 34], and can be easily expanded in vitro [33]. However, despite their highly differentiated phenotype, currently there are no data available on the regulation of telomerase and telomere length in primary human Treg under conditions of proliferation.

Using a broad spectrum of methods, our current report corroborates the observation published by Taams and colleagues [27] showing that Treg from healthy humans have shorter telomeres when compared to CD4⁺CD25⁻ T-cells. This is not surprising, as almost all Treg express CD45RO⁺ and therefore exhibit a memory phenotype, which is known to be associated with a decrease in telomere length when compared to CD45RA⁺ naive T-cells [32]. It is however noteworthy that telomere length of Treg isolated from patients suffering from epithelial cancer is preserved when compared to their age-matched counterparts from healthy individuals, despite their proven increase. It is of critical importance for the interpretation of these data that Treg from cancer patients have decreased levels of TREC when compared to Treg isolated from healthy age-matched controls. This observation supports the idea that these cells indeed underwent further post-thymic proliferation, rather than redistribution of Treg from secondary lymphoid organs into peripheral blood accounts for the observed increase. We cannot exclude that this may also reflect a lower thymic output of naive T-cells in cancer patients due to thymic involution, a process which has been shown in animal models of mammary tumors [1]. However, the observation that TREC content is comparable between the CD4⁺CD25⁻ fraction from healthy volunteers and cancer patients strongly argues for an active proliferation of the Treg pool. Thus, the preservation of telomere length might be due to the induction of telomerase activity in the expanded Treg population. Data from murine transplantation models would support this hypothesis. In fact, preservation of telomere length in T-cells from serially transplanted mice under conditions of clonal expansion was due to the induction of telomerase activity [2].

By analyzing the regulation of telomerase activity under conditions of in vitro expansion of isolated Treg, we could demonstrate an efficient induction of telomerase activity in proliferating Treg in vitro. Notably, the addition of exogenous IL-2 is required for both, Treg proliferation and induction of telomerase activity. The strict dependence of telomerase induction as well as hTert mRNA expression (data not shown) on the presence of IL-2 is in keeping with previous data showing that IL-2 represents a critical regulator of telomerase expression and activity in human peripheral lymphocytes [16, 17].

Using a sorting strategy based on the dilution of CFSE, we could further demonstrate for the first time that telomerase activity is primarily induced in the proliferating CFSE^low population during polyclonal in vitro Treg expansion, whereas the non-proliferating fraction did not exhibit significant telomerase activity. This is paralleled by an enhanced immuno-suppressive capacity of the expanded Tregs, which is in line with recent data [28] and fits also to the observation that telomerase activation is primarily induced in lymphocytes entering the cell cycle [6]. In contrast, a recent paper showed that non-proliferating lymphocytes are also capable to induce telomerase activity [13]. However, the ³H-Thy incorporation assay might be too insensitive for the definition of non-proliferating T-cells, thereby harboring the problem that a small proliferating T-cell population with high telomerase activity might explain the reported results.

Clonal expansion of T-cells has been associated with the induction of telomerase and subsequent maintenance of telomere length in vivo [2]. We observed a significant shortening of telomeres in proliferating Treg (i.e., the CFSE^low) in vitro, which differs to the data obtained from cancer patients showing a preservation of telomere length. Our data suggest that despite the rapid and efficient induction of telomerase activity in the proliferating part of Treg in vitro, telomere erosion cannot be avoided under in vitro growth conditions, which might be far more stimulatory than in vivo conditions leading to Treg expansion in cancer. Notably, the determination of the average telomere length represents only one parameter for the characterization of the “telomere status”. In addition, hTERT is also known to influence cell cycle properties independent of telomere maintenance, for example by regulating the cell survival of CD4⁺ T-cells upon overexpression of hTERT, demonstrating that hTERT expression is not necessarily correlated to maintenance of telomere length and genomic stability [21]. This is in line with our observation that despite high proliferation and telomerase activity, telomere length is shortened in vitro. Our data might be of relevance when considering the application of in vitro expanded Treg for the treatment of autoimmune diseases or GvHD. Since critical telomere shortening can be associated with genetic instability, in vitro rapidly expanded Tregs could be prone to neoplastic transformation in vivo.