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Immunology

Tumor-Induced Expansion of Regulatory T Cells by Conversion of CD4+CD25− Lymphocytes Is Thymus and Proliferation Independent

Barbara Valzasina, Silvia Piconese, Cristiana Guiducci and Mario P. Colombo
DOI: 10.1158/0008-5472.CAN-05-4217 Published April 2006

Abstract

The CD25− and CD25+ CD4 T-lymphocyte compartments are tightly regulated. We show here that tumors break such balance, increasing the number of CD4+CD25+ T cells in draining lymph node and spleen but not contralateral node of tumor-bearing mice. Tumor injection in thymectomized and CD25-depleted mice shows that CD4+CD25+ T-cell expansion occurs even in the absence of the thymus and independently from proliferation of preexisting CD25+ T cells. These newly generated cells are bona fide regulatory T cells (T reg) in terms of Foxp3 expression and suppression of CD3-stimulated or allogeneic effector cell proliferation. Transfer of congenic Thy1.1 CD4+CD25− T cells, from mice treated or not with vinblastine, into tumor-bearing or tumor-free mice and analysis of recovered donor lymphocytes indicate that conversion is the main mechanism for acquiring the expression of CD25 and Foxp3 through a process that does not require proliferation. Although conversion of CD4+CD25− T cells for generation of T regs has been described as a natural process that maintains peripheral T-reg population, this process is used by the tumor for immune escape. The prompt recovery of T regs from monoclonal antibody–mediated CD25 depletion in tumor-bearing mice suggests attempts able to inactivate rather than deplete them when treating existing tumors. (Cancer Res 2006; 66(8): 4488-95)

  • Regulatory T cells
  • Tumor immunology
  • CD4 T lymphocytes

Introduction

Naturally arising CD4+CD25+ regulatory T cells (T reg) originate in the thymus from high-avidity interactions of their T-cell receptors with self-peptide/MHC class II expressed on thymic stroma cells ( 1). They play an essential role in controlling T-cell numbers neutralizing autoreactive T cells ( 2). This subpopulation of CD4+ T cells is characterized by the expression of a member of the forkhead/winged-helix trascription factors, Foxp3 ( 3). To date, Foxp3 is the most specific T-reg marker; other surface molecules are preferentially expressed by T reg at the naive state while being largely expressed in activated CD4+ T lymphocytes: they include CTLA-4 ( 4), GITR ( 5, 6), OX40 ( 7), and CD103 ( 8). The role of T reg in controlling T-cell number has been documented in several studies. Foxp3−/− and CD25−/− mice completely lack T regs and suffer from uncontrolled T-cell expansion, which can be normalized by reconstituting the CD4+CD25+ cell compartment ( 9, 10). In addition, T-reg number is tightly regulated; irradiated, lymphopenic mice reconstituted with naive CD4+ T cells show a normal ratio of CD25− and CD25+ CD4 T cells, including T reg, even if an excess of T reg is cotransferred ( 11). Thus, T-reg number constantly ranges between 5% and 10% of the peripheral CD4+ T cells depending on the mouse strain.

As an exception to such tight control, T regs accumulate above the reference range in patients with carcinoma of breast or pancreas ( 12), with hepatocellular carcinoma ( 13), non–small cell lung cancer, and late-stage of ovarian cancer ( 14). Although in human, the increase of CD4+CD25+ T cells has been reported to be associated with the reduction of CD4+CD25− T cells ( 15), the origin and mechanism governing T-reg increase in cancer patients remains elusive. A very recent article shows proliferation of T reg in tumor-bearing mice and rats in response to transforming growth factor-β (TGF-β) released by tumor-recruited immature myeloid dendritic cells ( 16). However, proliferation alone cannot account for their large expansion and, moreover, does not explain the corresponding reduction of CD4+CD25− T cells. Several reports describe antibody-mediated depletion of T regs using anti-CD25 monoclonal antibody (mAb; refs. 17– 21) but no data are available on T-reg recovery in tumor bearers.

In this study, we analyzed T-reg number and function in tumor-bearing mice as well as their recovery after peripheral and central depletion by means of anti-CD25 mAb and thymectomy. We describe CD4+CD25− T-cell conversion into T regs as the main mechanism of T-reg replenishment and expansion in tumor-bearing mice.

Materials and Methods

Mice and treatments. BALB/c mice were purchased from Charles River (Calco, Italy). BALB/c Thy1.1 mice were kindly provided by H. Levitsky (John Hopkins University, Baltimore, MD) and were maintained under pathogen-free conditions in our animal facility according to institutional guidelines. Mice were used at 8 weeks of age, except thymectomy that was done at 5 weeks of age. In vivo treatment with anti-CD25 mAb (PC61 clone) was done using i.p. injection of 0.6 mg purified mAb from PC61 hybridoma 4 and 3 days before tumor administration. CT26 is a carcinogen-induced, undifferentiated colon carcinoma of BALB/c mice. Tumor cells were cultured in DMEM (Life Technologies, Paisley, United Kingdom) supplemented with 10% FCS (BioWhittaker, Walkersville, MD). Cells (5 × 104) were inoculated s.c. in naïve mice, and 2 × 105 cells were inoculated in thymectomized and PC61-treated mice. Vinblastine sulfate, 200 μg (VELBE, Crinos, Milan, Italy), was injected into the tail vein 15 hours before harvesting lymphocytes for adoptive transfer.

Purification of CD4+CD25+ and CD4+CD25− subsets. T cells were first enriched by passing the whole spleen on nylon wool columns (Polysciences, Warrington, PA). CD8+ cells were removed using anti-CD8 MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25+ cells were then separated from CD4+CD25− cells using the CD25+ T-cell isolation kit (Miltenyi Biotec) according to the instructions of the manufacturer. Flow cytometry showed that the separate fractions were >90% pure.

Antibodies and flow cytometric analysis. FITC-conjugated anti-CD4 (L3T4), phycoerythrin-conjugated anti-CD25 (PC61), FITC-conjugated anti-CD103 (M290), FITC-conjugated anti-CD45RB (C363-16A), phycoerythrin-Cy5-conjugated anti-CD4 (L3T4), and streptavidin-FITC were all purchased from BD Bioscience (San Diego, CA). FITC-conjugated anti-GITR (DTA-1), FITC-conjugated anti-CD62L (MEL-14), purified anti-OX40 (OX86), and APC-conjugated Thy1.1 (HIS51) were purchased from e-Bioscience (San Diego, CA). FITC-conjugated anti-CTLA-4 (1B8) was purchased from Southern Biotechnologies (Birmingham, AL). Antibodies were used at 5 μg/mL and staining was done in fluorescence-activated cell sorting (FACS) buffer (10% FCS in PBS) on ice for 45 minutes. FACS analyses were done on a FACScan (Becton Dickinson, Franklin Lakes, NJ). Intracellular staining of Foxp3 (FJK-16S) was done on purified CD4+ CD25+ according to the instructions of the manufacturer (e-Bioscience). TGF-β1 production was measured by a specific ELISA according to the instructions of the manufacturer (Bender Medsystems, Vienna, Austria).

Carboxyfluorescein succinimidyl ester labeling. For in vitro assays, purified CD4+CD25- were labeled by incubation with 2 μmol/L carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) in PBS containing 5% fetal bovine serum (FBS) for 5 minutes at 37°C. Cells were then washed twice with PBS and used in vitro for proliferation assay. For in vivo experiments, purified CD4+CD25− cells were incubated (1 × 107/mL) with 5 μmol/L CFSE in PBS containing 5% FBS for 15 minutes at 37°C and then washed twice with PBS before in vivo injection into mice.

In vitro suppression assays. To test T-reg suppressive activity, 5 × 104 CD4+CD25− cells were cultured with 5 × 104 accessory cells (consisting in the whole spleen irradiated with 3 Gy) with or without T regs for 72 hours in complete medium containing RPMI 1640 (Sigma, St. Louis, MO) supplemented with 5% FCS, 2 mmol/L l-glutamine, 200 units penicillin, and 200 mg/mL streptomycin (Sigma). For stimulation, 1 μg/mL anti-CD3 (e-Bioscience) or 5 × 104 allogeneic irradiated splenocytes were added. [3H]thymidine (1 μCi/well; Amersham, Piscataway, NJ) was added for the last 10 hours of culture.

To distinguish CD4+CD25− cell proliferation from any contaminating proliferating cells, CD4+CD25− were labeled with CFSE assayed in triplicates by flow cytometry.

In vivo conversion assay. To study in vivo conversion, Thy1.1-derived CD4+CD25− were labeled with 5 μmol/L CFSE for 15 minutes at 37°C. Ten million cells were transferred into recipient mice by tail vein injection when tumor diameter reached 2 to 3 mm. Percentage of converted CD4+CD25+ in lymph node and spleens was assessed after 10 days. Cells were stained with anti-CD25 phycoerythrin, phycoerythrin-Cy5 anti-CD4, and APC-Thy1.1 antibodies. The percentage of CD25+ cells over CFSE+ cells was calculated on gated Thy1.1+ CD4+ cells.

Foxp3 mRNA analysis. Approximately 5 × 106 CD4+CD25− or CD4+CD25+ cells were purified from spleen and lymph node of tumor-bearing or naïve mice. Total RNA was extracted with TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was synthesized from the RNA using oligo(dT) and M-MLV Reverse Transcriptase (Life Technologies). Primers to detect the Foxp3 cDNA by PCR were 5′-CAGCTGCCTACAGTGCCCCTAG-3′ and 5′-CATTTG CCAGCAGTGGGTAG-3′. Housekeeping control gene was β2-microglobulin. β2-microglobulin primers were 5′-ATGGCTCGCTCG GTGACCCTAG-3′ and 5′-TCATGATGCTTGATCACATGTCTCG-3′. All PCR analyses were done with a Taq polymerase (Promega, Madison, WI) using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) for 25 cycles.

Statistical analysis. Data were analyzed using a two-sided Student's t test. All analyses were done using Prism software (GraphPad Software, San Diego, CA). Differences were considered significant at P < 0.05.

Results

Increase in CD4+CD25+ cells in spleen and draining lymph nodes of tumor-bearing mice. Although several groups reported accumulation of CD4+CD25+ T regs inside the tumor ( 15, 22– 24), no compelling investigations are available on T-reg expansion in secondary lymphoid organs in the mouse system. Using the CT26 colon carcinoma, we found that although few lymphocytes infiltrate the tumor (<0.5% of the entire tumor cellularity), almost all recovered CD4+ T cells bear the CD25 marker ( Fig. 1A ). To investigate whether tumor recruitment of CD4+CD25+ T cells corresponds to a drop in secondary lymphoid organs, we evaluated the content of CD4+CD25+ T cells in the spleen, tumor-draining lymph nodes, and contralateral lymph nodes. Spleen and draining lymph nodes showed increased number of CD4+CD25+ T cells, whereas the contralateral lymph nodes had the same cell number of tumor-free mice ( Fig. 1B-D). Such CD4+CD25+ T-cell expansion was confirmed in two other murine tumors, namely the TSA and 4T1 mammary carcinomas ( Fig. 1E-F) and seems to correlate with tumor size (data not shown). The CD4+CD25+ T cells described above are bona fide T regs on the basis of Foxp3 expression analyzed at mRNA ( Fig. 2A ) and protein levels ( Fig. 2B). Although the majority of CD4+CD25+ T cells are also Foxp3 positive, a small population of cells express little or no Foxp3. Functional assay confirmed that CD4+CD25+ T cells purified from tumor, lymph node, and spleen of tumor-bearing mice inhibit CFSE-labeled CD4+CD25− T-cell proliferation to the same extent of T regs purified from tumor-free mice ( Fig. 2C). The same T reg–mediated inhibition against allogeneic stimulators or anti-CD3-stimulated effector cells was obtained when measured as thymidine incorporation instead of CFSE dilution (data not shown).

Figure 1.
Figure 1.

Increase of CD4+CD25+ T cells in tumor-bearing mice. A, CD4+ T cells purified from TIL; B, contralateral lymph node (CLN), and draining lymph node (DLN) of CT26-bearing mice were analyzed by flow cytometry for expression of CD4 and CD25. Comparison between the percentages of CD25+ cells among the CD4+ T cells from contralateral lymph nodes and draining lymph nodes of mice bearing CT26 (C), TSA (E), or 4T1 (F) tumors and from spleen of CT26-bearing and tumor-free mice (D) assessed by flow cytometry. Pooled data from three independent experiments. Each dot corresponds to a single mouse. Solid line, median value. ***, P < 0.005.

Figure 2.
Figure 2.

CD4+CD25+ T cells from tumor-bearing mice show similar Foxp3 expression and in vitro suppressive activity of naturally arising T regs. A, RT-PCR analysis of Foxp3 expression on mRNA isolated from CD4+CD25+ T cells purified from spleen of tumor-free (SPL TF) and tumor-bearing mice (SPL-TB), or draining lymph node of tumor-bearing mice (DLN-TB). Results obtained after 25 cycles are shown because more cycles allow detection of Foxp3 expression even in CD4+CD25− cells in accordance with the small fraction of cells also CD45RBlow that express it (see Discussion). B, intracellular Foxp3 expression (solid histogram) in CD4+CD25− (CD25−) and CD4+CD25+ T cells (CD25+) purified from draining lymph node, spleens of tumor-bearing and tumor-free mice, as well as total CD4+ T cells from TILs. Thin line, isotype control rat IgG2a. One of four independent experiments showing similar results. C, CFSE-labeled CD4+CD25− T cells (1 × 105) were seeded with CD4+CD25+ T cells (1 × 105) in the presence of accessory cells (1 × 105) and 1 μg/mL α-CD3 antibody. After 72 hours, cells were collected and analyzed by flow cytometry. Percentage of proliferating cells ± SD of pooled data from three independent experiments is given along with one representative profile.

CD4+CD25+ T cells can be generated in secondary lymphoid organs in a thymus-independent way. To test whether the increased number of T regs in secondary lymphoid organs of tumor-bearing mice results from expansion of preexisting or from newly generated T regs, we inoculated tumor cells in mice previously depleted of CD4+CD25+ cells by anti-CD25 mAb (PC61 hybridoma) and compared the number of CD4+CD25+ T cells in mice bearing or not bearing CT26 tumors. To avoid the 30% of tumor rejection that occurs in this setting because of T-reg depletion ( 17), we doubled the dose of CT26 cells. Spleen, draining lymph nodes, contralateral lymph nodes, and tumor-infiltrating lymphocytes (TIL) were collected when the tumor size was ∼7 to 8 mm. Figure 3A shows that although CD25+ cells were initially depleted, their replenishment was more vigorous in spleen and draining lymph nodes, which had twice the number of T regs than contralateral lymph nodes or PC61-treated tumor-free mice ( Fig. 3A and B).

Figure 3.
Figure 3.

Repopulating CD4+CD25+ T cells in lymph node, spleen, and thymus of tumor-bearing and tumor-free mice depleted of CD25. BALB/c mice were injected i.p. with 0.6 mg α-CD25 mAb at days −4 and −3. At day 0, they were inoculated (TB) or not (TF) with 1 × 105 CT26 cells. When tumors reached 8 mm, lymph node (LN), spleen, and thymus were collected. Percentage of CD4+CD25+ T cells among CD4+ T cells in lymph node (A), spleen (B), and thymus (C) of tumor-bearing and tumor-free mice was assessed by flow cytometry. Pooled data from three independent experiments. Each dot corresponds to a single mouse. Solid line, median value. ***, P < 0.005.

Because T regs originate in the thymus, we compared their number in the thymus of tumor-bearing mice and tumor-free mice. In sharp contrast to what has been observed in the spleen and draining lymph nodes, the percentage of thymic CD4+CD25+ T cells did not differ between tumor-bearing and tumor-free mice ( Fig. 3C); this suggests the peripheral origin of newly formed CD4+CD25+ T cells. To confirm that the thymus is not involved in tumor-induced T-reg regeneration and to further distinguish between de novo generated and preexisting T regs, mice were thymectomized (Tx) and depleted of T regs by means of an antibody to CD25 (Tx-CD25) before injecting the tumor ( Fig. 4A ). Thymectomy before antibody depletion avoids repopulation by thymus-derived CD4+CD25+ T cells. Twenty days after Tx-CD25 treatment, the percentage of T regs in lymph node and spleen of tumor-free mice was 0.7 and 2.5 of the total CD4+ T cells, respectively, whereas in tumor-bearing mice they were 5- and 2-fold more numerous (i.e., 3.5% and 5% in lymph node and in spleen, respectively; Fig. 4B and D). Also, the majority of the CD4 T cells present in the TIL (>90%) are also CD25 positive ( Fig. 4C).

Figure 4.
Figure 4.

Tumor-induced expansion of CD4+CD25+ T cells occurs in the periphery in the absence of preexisting T regs. BALB/c mice were thymectomized at the day −7 and injected i.p. with 0.6 mg of α-CD25 mAb at days −4 and −3. At day 0, they were inoculated or not with 2 × 105 CT26 cells. When tumors reached 8 mm, lymph node, spleen, and thymus were collected. A, effective T-reg depletion at time of tumor injection. No CD4+CD25+ T cells were present in lymph nodes as shown in a representative dot plot. B, representative dot plots for CD4 and CD25 expression in lymph nodes of Tx-CD25-depleted mice bearing (right) or not bearing the tumor (left, draining lymph node); the percentage of CD4+CD25− and CD4+CD25+ T cells among total cells are indicated. C, representative dot plot of CD4 and CD25 staining done on TILs of TB-Tx-CD25 mouse. Percentage of CD4+CD25+ T cells among total cells is reported. D, percentages of CD25+ cells among the total CD4+ T cells analyzed in (B). E, percentage of CD4+CD25− T cells among the total T-cell population from the same mice. F, percentages of CD25+ cells among the total CD4+ T cells in spleens collected from tumor-bearing and tumor-free mice. Pooled data from two independent experiments. Each dot corresponds to a single mouse. Solid line, median value. ***, P < 0.005.

Although T regs can be generated in the absence of thymus from peripheral conversion of CD4+CD25− T cells ( 25, 26), our data indicate that in tumor-bearing mice, this process is forced and corresponds to an increase in the percentage of CD4+CD25+ within the CD4+ T-cell compartment mostly because of a reduction of CD4+CD25− T cells (see, e.g., the percentage of CD4+CD25− T cells in Fig. 4B and E).

Newly formed CD4+CD25+ are phenotypically and functionally indistinguishable from naturally occurring T regs. Newly generated T regs from CD25 depleted and thymectomized tumor-bearing and tumor-free mice were compared. Cytofluorimetric analysis and reverse transcription-PCR (RT-PCR) showed that CD4+CD25+ cells purified from TIL, spleens, and draining lymph nodes of tumor-bearing and from spleens of tumor-free mice express Foxp3 ( Fig. 5A and data not shown). Other surface markers associated with naturally occurring T regs were analyzed by FACS. GITR, CTLA-4, OX40, CD45RB, and CD62L molecules take part to T-reg function ( 4, 5, 7, 27– 29), whereas CD103 mostly marks effector/memory T regs ( 8). The profile of tumor-bearing-derived CD4+CD25+ T cells purified from draining lymph node and spleen was roughly similar to that of naturally occurring T regs from spleen of tumor-free mice ( Fig. 5). Although highly expressed, the level of GITR, CTLA-4, and OX40 were further increased in tumor-bearing versus tumor-free mice, whereas the expression of CD45RB molecule decreased. Reduction of CD62L coupled with increased CD103 expression may suggest that more T regs with effector/memory phenotype populate tumor-bearing mice.

Figure 5.
Figure 5.

Foxp3 and surface marker expression is similar between T regs from tumor-free mice and from Tx-CD25− tumor-bearing mice. A, BALB/c mice were thymectomized at the day −7, injected i.p. with 0.6 mg α-CD25 mAb at days −4 and −3. At day 0, they were inoculated with 2 × 105 CT26 cells. When the tumor reached 8 mm, lymph nodes and spleens were collected, and CD4+CD25− and CD4+CD25+ T cells were purified and analyzed for the expression of Foxp3. B, same experiments as described in (A) were done to evaluate the expression of GITR, CTLA-4, OX40, CD45RB, CD62L, and CD103 (filled histogram) on CD4+CD25+ T cells (thin line, isotype control). CD4+CD25+ T cells purified from spleen of tumor-free mice (TF) were used for comparison. One representative of two independent experiments is shown.

To test the activity of the newly generated T regs, CD4+CD25+ or CD4+CD25− cells from tumor-bearing mice were added to CFSE-labeled CD4+CD25− effector cells from naive mice. The total number of effector T cells that divided in presence of CD4+CD25+ was compared with that obtained in presence of CD4+CD25− T cells that might have other regulatory or competing activity ( 30). In these experiments, we used as control of effective inhibition CD4+CD25+ T cells derived from spleens of naive mice rather than CD4+CD25+ from Tx-CD25 tumor-free mice because their yield was insufficient for purification. As reported in Fig. 6 , tumor-bearing-derived CD4+CD25+ T cells suppressed anti-CD3-mediated proliferation of effector T cells to the same extent of naive T regs. Taken together, these data indicate that CD4+CD25+ T cells induced by the tumor in the absence of thymus posses all the features of naturally occurring T regs.

Figure 6.
Figure 6.

Tumor-converted CD4+CD25+ T cells suppresses in vitro proliferation of effector T cells. A, CD4+CD25+ T cells (1 × 105) purified from spleens and draining lymph node of tumor-bearing mice were added at 1:1 ratio to CFSE-labeled CD4+CD25− effector T cells purified from spleens of naive mice with irradiated spleen cells (1 × 105, accessory cells) as stimulator in the presence of 1 μg/mL α-CD3 antibody. As control, effector T cells were cultured in the absence or presence of CD4+CD25− T cells (1 × 105). Three days later, cultures were analyzed by cytofluorimetric analysis and the total number of effector cells that done one or more division was determinate by CFSE dilution. The percentage of suppression was evaluated as the total number of effector T cells that divided in presence of CD4+CD25+ and compared with nonsuppressive but competing CD4+CD25− T cells. B, CD4+CD25− T cells were seeded at the indicated ratio with CD4+CD25+ or CD4+CD25− T cells purified from spleen or draining lymph node of tumor-free or tumor-bearing mice in the presence of accessory cells and 1 μg/mL α-CD3 mAb, proliferation was evaluated after 3 days by thymidine incorporation assay. One representative of two independent experiments is shown.

Tumor enhances peripheral conversion of CD4+CD25− T cells into T regs. The homeostatic control of T-cell number requires a constant amount of T regs. This implies that when CD4+CD25− T cells are transferred into naive mice, a fraction of them should convert into CD4+CD25+ T cells to maintain the steady-state proportion of CD4+CD25+/CD4+CD25− T cells ( 26, 30). Such demonstration of CD4+CD25− conversion into T regs and our observation that an increased T-reg number corresponds to a reduced number of CD4+CD25− in Tx-CD25-treated tumor-bearing mice ( Fig. 4A) prompted us to investigate whether conversion takes part to T-reg expansion in tumor-bearing mice. To this end, CD4+CD25− purified from lymph node and spleen of Thy1.1 congenic mice were labeled with CFSE and inoculated into mice bearing or not bearing tumors of 2 to 3 mm in size ( Fig. 7A ). When the tumor reached 7 to 8 mm (∼10 days after Thy1.1+ CD4+CD25− transfer), lymph nodes, spleens, and TILs were collected, and Thy1.1+CD4+ donor lymphocytes ( Fig. 7B, left) were analyzed for expression of CD25 as function of conversion and for CFSE dilution as function of proliferation ( Fig. 7B, right). In this system, the CD4+CD25− T cells undergo conversion apparently in the absence of marked proliferation. Indeed, the cumulative results show that in draining lymph node and spleens ( Fig. 7D) of tumor-bearing mice, the percentage of CD4+CD25− cells that convert into CD4+CD25+ is almost double than that observed in tumor-free mice (7% versus 3%, respectively). In the TIL population, the majority of the donor Thy1.1 CD4+CD25− T cells were collected as CD4+CD25+ T cells ( Fig. 7C). Despite the different rate of conversion, CD4+CD25+ T cells converted from donor CD4+CD25−, in both tumor-free and tumor-bearing mice, were also Foxp3 positive when gated on Thy1.1. This confirms their T-reg phenotype ( Fig. 7F).

Figure 7.
Figure 7.

Tumor-induced T regs derive from peripheral conversion of CD4+CD25− T cells. A, characterization of donor CD4+CD25− T cells before transfer. CD4+CD25− T cells account for 12% of total lymph node before purification (left); they were purified up to 99.8% before transfer (right). B, conversion of donor CD4+CD25− T cells into CD25+ upon in vivo transfer. Purified Thy1.1+ CD4+CD25− T cells (10 × 106) were transferred into BALB/c mice bearing or not a 2 to 3 mm tumor. After 10 days, lymph node and spleens were collected and labeled with Thy1.1, CD4 and CD25; Thy1.1+ CD4+ cells were gated (left) and analyzed by FACS for CD25 and CFSE expression (right). Representative dot plots done on draining lymph node are shown. C, donor cells collected from tumor. Representative dot plot for the expression of CFSE and CD25 in TIL of tumor-bearing recipient is shown. D, cumulative data of the Thy1.1+ CFSEhigh cells conversion in lymph node (left) and spleens (right) cells is reported. Each dot represents a single mouse. Solid line, median value. ***, P < 0.005. E, converted CD4+CD25+ T cells express Foxp3. Total CD4+CD25+ T cells were purified from lymph node of recipients tumor-free or tumor-bearing mice and analyzed by FACS for expression of Thy1.1. Thy1.1-positive cells were further analyzed for expression of Foxp3 (filled histogram), percentages of Foxp3+ cells are indicated, thin line represents isotype control (rat IgG2a). F, inhibition of T-cell proliferation does not alter conversion. The same experiment described in (B) was repeated after pretreating Thy1.1 mice with vinblastine sulfate for 15 hours before collecting spleen and LN. Percentage of converted cells is reported as Thy1.1+/CD4+/CD25+ and CFSEhigh. Cumulative data of converted cells in draining lymph nodes, contralateral lymph nodes, and lymph nodes of tumor-free mice (left) and spleens (right) cells are shown. Each dot corresponds to a single mouse; solid line indicates the median value. ***, P < 0.005.

To confirm that conversion does not require cell proliferation, donor Thy1.1+ mice were pretreated with vinblastine 15 hours before harvesting lymph node and spleen for T-cell transfer ( 31). Purified CD4+CD25− were labeled with CFSE and injected into tumor-bearing or tumor-free mice as above. Even in the absence of proliferation, the amount of CD4+CD25− T cells that converted into T regs was double in tumor-bearing mice than in tumor-free mice ( Fig. 7E).

The data show that T-reg expansion occurring in tumor-bearing mice is mainly due to conversion from CD4+CD25− T cell.

Discussion

Originally thought of restricted thymic origin ( 2), recent evidences indicate that T regs can also be generated in the periphery. In adult mice, their depletion by means of the anti-CD25 mAb and thymectomy is followed by complete reconstitution within 48 days ( 25). In addition, purified CD4+CD25− T cells produce a fraction of CD4+CD25+ T cells when transferred into RAG−/− mice ( 32). CD4+CD25− cells expressing Foxp3 have been described as a peripheral reservoir of T cells that promptly convert into the CD4+CD25+ pool to replenish homeostatic condition in repopulated RAG−/− mice ( 26). When transferred into naive mice, a fixed proportion of CD4+CD25− T cells can convert into CD4+CD25+ T cells to maintain constant their ratio ( 30). Low or excessive number of T regs characterize pathologic conditions like autoimmunity and cancer, respectively. In cancer, T-reg depletion has been tested either alone ( 17– 19) or in combination with vaccination ( 21, 33, 34) to prevent or treat transplanted tumors. However, reconstitution after depletion has never been studied in tumor bearers. In this article, we provide the first comprehensive analysis of the excessive conversion of CD4+CD25− T cells into T regs occurring in tumor-bearing mice.

We show that tumors of different origins induce an increase in T-reg number over the homeostatic condition, in tumor-draining lymph nodes and spleen but not in contralateral lymph nodes early after T-reg depletion in a thymus-independent way. Using ovalbumin or hemagglutinin transgenic lymphocytes, several studies showed that T regs can be generated both in vitro and in vivo by CD4+CD25− T-cell conversion ( 35, 36). A single report confirmed the phenomenon in a nonlymphopenic environment and using non-T-cell receptor transgenic lymphocytes ( 30). Under such physiologic condition, but in the presence of the tumor, we show that in draining lymph node and spleen, the number of CD4+CD25− T cells that convert into CD4+CD25+ is double than that converting in contralateral lymph nodes or in lymph node and spleen of tumor-free mice. This conversion does not require cell proliferation, whereas converted cells might cycle thereafter. The negligible difference between the number of T regs converted in mice receiving Thy1.1+ donor, treated or not with vinblastine, speaks in favor of conversion as the main mechanism of T-reg expansion, at least at the tested time point (∼10 days after CD4+CD25− cells transfer).

By studying CD4+CD25+ T-cell regeneration in thymectomized and CD25-depleted mice, either tumor-bearing or tumor-free, we established a system in which the majority of the CD4+CD25+ T cells recovered derive from conversion, thus excluding proliferation of preexisting T regs. In this system, purified CD4+CD25+ from draining lymph node and spleen of tumor-bearing mice suppress the proliferation of effector T cells and express high levels of Foxp3 at mRNA and protein levels, indistinguishable from natural arising T regs. Their cell surface phenotype slightly differs from naturally occurring T regs for the expression of CD62L and CD103, suggesting a different distribution between lymphoid organs and peripheral tissues. Tumor-bearing-derived T regs express ∼30% lower and higher level of CD62L and CD103, respectively, than T regs from tumor-free mice. Although T regs in vitro suppress effector T-cell proliferation regardless of their level of CD62L expression, in vivo only CD62Lhigh cells have the ability to suppress diabetes ( 37) or graft-versus-host disease ( 38). Such discrepancy is also described for CD103 ( 8). CD103high and CD103low T regs are characterized by a different proportion of cells expressing CD62Lhigh. Functionally, CD103high T regs suppress in peripheral tissues and are predominantly CD62Llow. CD103low T regs are in majority CD62Lhigh and remain within secondary lymphoid organs ( 39). Accordingly, a correlation between CD62L and CCR7 expression on T regs has been described. T regs expressing CCR7 and CD62Lhigh migrate in response to lymphoid chemokines, whereas those that are CD62Llow express CCR2 and CCR4 and preferentially migrate toward inflammatory chemokines ( 29). Among TILs, the majority of CD4+ T cells are also CD25 positive and express CD103 (not shown). A 50% more CD103high CD4+CD25+ T cells are collected from draining lymph node and spleen of tumor-bearing thymectomized mice than in tumor-free mice. Although these data might confirm the existence of different subpopulations of T regs ( 39, 40), in our view, they may suggest active circulation of T regs toward systemic colonization.

Tumor creates environment similar to chronic inflammation and is a source of both chemokines and TGF-β ( 41, 42) that influence migration and conversion of CD4+ T cells, respectively ( 43– 45). TGF-β induces T-reg proliferation and tumors not secreting TGF-β can directly license immature myeloid dendritic cells to produce it through unknown mechanisms ( 16). The three tumors used in this study, plus all spontaneous mammary carcinoma derived from BALB/c NeuT mice ( 46) tested thus far, produce TGF-β (refs. 47, 48 and data not shown). According to the general finding describing TGF-β as one of the most potent tumor produced suppressive factor and its already described role in T-reg conversion, it is very likely that TGF-β has a main role in promoting CD25− to CD25+ conversion in tumor bearers. In addition, a very recent article shows that in the presence of suboptimal dose of antigen and suboptimal dendritic cell activation, a situation resembling a tumor setting, TGF-β enables conversion of naïve T cells into suppressor cells while inhibiting proliferation of preexisting T regs ( 49). This process might advantage tumor escape by (a) inhibiting proliferation of existing T regs that are maintained through a continuous interaction with dendritic cells, providing self-antigen and costimulation, and (b) favoring generation of new T regs, from the broader, although less avid, T-cell receptor repertoire of circulating CD4+CD25− T cells, which suppress in a nonantigen-specific manner ( 2). Further experiments transferring CD4+CD25− T cells derived from dominant negative TGF-β receptor type II (dnTGF-βRII) mice ( 50) into tumor-bearing mice will prove the role of this cytokine in tumor-induced conversion of naïve T cells into T regs.

The connection between a tumor and its draining lymph node establishes whether activation or suppression of the immune response will occur. All our experiments indicate that nondraining lymph node of tumor bearers have phenotype and function almost identical to that of tumor-free mice, at least for type and size of tumors analyzed. In accordance with the CD103 and CD62L expression observed in those setting, it might be possible that contralateral lymph nodes will also be invaded by T regs as long as tumor progresses.

Recently, it has been shown that in homeostatic condition, a minority of CD4+CD25− CD45RBlow T cells express Foxp3 and undergo conversion ( 26). We observed that all the CD4+CD25+ isolated from Tx-CD25-depleted tumor-bearing mice express low level of CD45RB and therefore we can speculate that, similarly to what happens in tumor-free mice, the population of CD25− T cells that convert into T regs encompassed the CD45RBlow subset.

Two articles using different tumor models described that T regs inhibit the immune response predominantly at the tumor site and that T regs preferentially accumulate in the tumors, whereas few remain inside the draining lymph node ( 51, 52). Contrary to such observations and in agreement with several others ( 12, 13, 15, 23), we observed increased T-reg number in draining lymph node and spleen of tumor-bearing mice using three different tumor types. The T-reg phenotype we have observed in draining lymph nodes and TILs of Tx-CD25-depleted mice suggests that different subsets of T regs, characterized by different expression of CD62L and CD103, are actively circulating throughout tissues and secondary lymphoid organs.

In conclusion, our data showing CD4 T-cell conversion as the major mechanism of T-reg expansion and showing early recovery of T regs in tumor bearers indicate the need of inhibiting rather than depleting T regs toward immunotherapy of established tumors, a process not impossible if acting through selected molecules like GITR ( 53) or OX40 ( 7).

Acknowledgments

Grant support: Associazione Italiana per la Ricerca sul Cancro, Fondo per gli Investimenti di Base (FIRB code RBNE017B4C) and Fellowships from Federazione Italiana per la Ricerca sul Cancro (B. Valzasina and S. Piconese).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Ivano Arioli for technical assistance.

Footnotes

  • Note: B. Valzasina and S. Piconese contributed equally to this work.

  • Received November 28, 2005.
  • Revision received February 2, 2006.
  • Accepted February 10, 2006.

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Cancer Research: 66 (8)
April 2006
Volume 66, Issue 8

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Tumor-Induced Expansion of Regulatory T Cells by Conversion of CD4+CD25− Lymphocytes Is Thymus and Proliferation Independent
Barbara Valzasina, Silvia Piconese, Cristiana Guiducci and Mario P. Colombo
Cancer Res April 15 2006 (66) (8) 4488-4495; DOI: 10.1158/0008-5472.CAN-05-4217
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