Outgrowth of CD4^low/negCD25^int T cells with suppressor function in CD4⁺CD25⁺ T cell cultures upon polyclonal stimulation ex vivo

Abstract

CD4⁺CD25⁺ regulatory T cells play an essential role in controlling autoimmunity and allograft rejection. Several ex vivo activation and expansion protocols have been developed to amplify cell numbers and suppressor function of murine and human Tregs. We demonstrate here that ex vivo activation and expansion of murine Tregs resulted in an enrichment of a CD4^low/negCD25^int T cell population that was more than 20 fold more potent than expanded conventional Tregs in suppressing an in vitro CD4⁺CD25⁻ T cell response to allo-antigen. The generation of CD4^low/negCD25^int T cells was independent of the presence of Tregs in the culture and suppressor function was acquired only after activation and expansion. CD4^low/negCD25^int T cells expressed either an αβ or γδ T cell receptor, had an activated phenotype and did not express the transcription factor FoxP3. Despite expressing the cell surface antigens lymphocyte activation gene (LAG)-3 (CD223) and CD103, neither were essential for suppressor cell function. Suppression by CD4^low/negCD25^int T cells was prevented by a semi-permeable membrane and was independent of IL-10 and TGF-β. In summary, we describe here CD4^low/negCD25^int FoxP3^neg T cells with highly potent suppressor cell function derived from cultures of an enriched population of CD4⁺CD25⁺ T cells that may contribute to the suppressor activity of ex vivo expanded bone fide Treg cells.

Introduction

CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) have been shown to play an essential role in regulating peripheral tolerance in mice and humans (1–4). Naturally occurring Tregs comprise 5–10% of peripheral mouse CD4⁺ T cells and 2–5% of peripheral human blood CD4⁺ T cells. Isolation of fresh Tregs would likely result in insufficient numbers of cells for many clinical applications. Because previously activated Tregs have a higher suppression capacity compared to fresh cells and due to limitations in fresh Treg cell numbers that can be acquired, several ex vivo activation and expansion protocols have been developed for both mouse and human Tregs. Such methods most often include polyclonal activation using antibodies to the TCR and costimulatory molecules bound to a plastic surface or inert beads (5–9). Antigen-specific activation and expansion methods have also been established using antigen-pulsed or allogeneic APCs (10–12).

Tregs used for expansion are most often isolated based upon CD25 expression. However CD25 is also expressed on other cell types and its expression pattern is not identical to the specific Treg transcription factor FoxP3. Moreover, Tregs are naturally anergic and have a growth disadvantage compared to conventional T cells that also express CD25. The T cell growth factor IL-2 can partially overcome this defect in proliferation and is thus required for Treg expansion. IL-2 is also a growth factor for other T cells and lymphocytes including NK cells and γδ T cells which express the IL-2R. As such, the final outgrowth of T cells from IL-2 supported cultures initiated with enriched naturally occurring Tregs may be substantially affected by “contaminating” non-Treg cells, especially in situations in which FACS sorting is not used. In this study we evaluated the phenotype and function of T cells generated in αCD3 mAb plus IL-2 supported expansion cultures initiated with murine Tregs obtained by a negative selection to obtain CD4⁺ T cells followed by a positive selection for CD25 using magnetic beads. We discovered the outgrowth of a very potent suppressor population that was CD3⁺CD4^low/negTCR⁺CD25^intFoxP3^neg cells. This T cell population shared qualities with Tregs in terms in their mechanisms of suppression and some surface molecules. However, overall phenotypically they resembled activated T cells and secreted cytokines released by Th1 type cells. Within this CD3⁺CD4^low/negTCR⁺CD25^int T cell population we detected CD8, γδ and NKT cells that all contained suppressor function and were similar in their activation status and expression of surface and intracellular molecules. Our findings suggest that polyclonal activation and expansion of Tregs in the presence of IL-2 can lead to an enrichment of other T cell populations that acquire regulatory function and can suppress effector responses to allo-antigen.

Material and Methods

Mice

C57BL/6 (B6), B6.Ly5.2 (CD45.1⁺) and BALB/c mice were purchased from the National Institutes of Health. B6.C-H2^bm12/KhEg and B6.129 P2-IL10^tm/Cgn (IL-10^−/−) mice were purchased from The Jackson Laboratory. B6.LAG-3 deficient (LAG-3^−/−) mice were generated as described (13). All mice were housed in a specific pathogen-free facility in micro-isolator cages according to the NIH guidelines.

Cell Purification

Axillary, mesenteric, sacral and inguinal lymph nodes were collected from 6–12 week old female mice into PBS containing 2% FCS (Hyclone). CD4⁺ T cells were isolated as described previously (14). The purity of the preparation was determined by FACS to be at least 95% CD4⁺ T cells. To enrich for CD4⁺CD25⁺ T cells, purified CD4⁺ T cells were incubated with anti-CD25 biotin (7D4), followed by streptavidin-PE; (both from Pharmingen) or anti-CD25 PE (PC61). After incubation with MACS anti-PE MicroBeads, cells were positively selected on MS or LS MACS separation columns (both Miltenyi Biotec). Column separation was repeated until CD25 purity was at least 98%. CD4⁺CD25⁻ cells were used as responder cells in suppression assays. Similarly, separation of expanded CD4⁺CD25⁺ from CD4^low/negCD25^int cells from expanded cultures was performed using an anti-CD4 PE mAb (RM4-5). Purity of both populations was over 99%. For separation of fresh CD4⁺CD25⁺ and CD4^low/negCD25^int cells, anti-CD4 CyChrome mAb (RM4-5) was used to label cells, followed by sorting on a FACS DIVA or FACS Aria. Purity of sorted cell populations was over 99%. For some experiments expanded cells were stained with anti-CD4 CyChrome mAb and anti-LAG-3 PE mAb or anti-CD103 PE mAb, followed by sorting on a FACS Aria.

CD4⁺CD25⁺ in vitro activation and expansion

Enriched CD4⁺CD25⁺ cells were suspended at a final concentration of 0.3 – 0.7 × 10⁶ cells/ml in DMEM complete media and cultured in 24- or 48-well plates (Costar) (15). CD4⁺CD25⁺ cells were activated for 3 days with 0.5 µg/ml plate-bound (pb) anti-CD3ε mAb (145-2C11). Cultures were maintained at 0.5–2 × 10⁶ cells/ml and supplemented with 100 U/ml human rIL-2 (Amgen) throughout the culture period every 2–3 days.

In vitro suppression assay

Freshly isolated CD4⁺CD25⁻ T cells were mixed with T cell depleted irradiated (30 Gy) bm12 splenocytes (as previously described) at a 1:1 ratio at a final concentration of 1.5 ×10⁶ total cells/ml in DMEM complete media (14). Suppressor cells were added at various concentrations as indicated. Cell cultures were incubated at 37°C and 10% CO₂ in 200 µl/well in 96 well plates (Costar) in at least triplicates. After 6 days of culture cells were pulsed with tritiated thymidine (1 µCi/well) (Amersham Life Sciences) for 16–18 hours, the cells were harvested, and tritiated thymidine uptake was assessed using a gas-operated β–plate reader (Packard Instrument Company). 100 µg/ml anti-TGF-β mAb (clone 1D11.16.8, mouse IgG1, American Type Culture Collection) was added to suppression assays using IL-10^−/− T cells. For suppression assays including transwell membranes CD4⁺CD25⁻ cells were mixed one to one with irradiated T cell depleted bm12 splenocytes at a concentration of 3×10⁶ cells/ml and cultured in the bottom well (600 µl/well) of 24 well transwell plates (Costar). Suppressor cells were adjusted to a concentration of 10×10⁶ cells/ml and cultured in the transwell insert (100 µl/insert) of 24 well plates. Tritiated thymidine was added on day 6 and after 16–18 hours cells from the bottom wells were transferred into 96 well plates, harvested and tritiated thymidine uptake assessed.

Flow cytometry and Antibodies

Mouse-specific antibodies were purchased from BD Pharmingen or eBioscience and staining was performed according to manufacture's protocol. Anti-granzyme B mAb was purchased from Caltag Laboratories). Acquisition was performed using a FACScalibur (BD Bioscience) and data were analyzed using FlowJo software (Tree Star Inc.).

Surface and intracellular cytokine staining

Cells were harvested and plated on anti-CD3 mAb coated 96-well plates at a concentration of 5–10×10⁶ cells/ml for 4–5 h in the presence of IL-2 and monensin at 37°C and 10% CO₂. Surface staining was performed for 20 min. Cells were fixed with Fixation/Permeabilization buffer (BD Pharmingen) for 20 min and washed twice with Permeabilization buffer (BD Pharmingen) and analyzed by FACS.

Results

In vitro activation and expansion of bead isolated Tregs results in an enrichment of a potent CD4^low/negCD25^int suppressor population that is independent from CD4⁺CD25⁺ T cells

We used an established protocol for isolation of Tregs consisting of a negative selection method for CD4⁺ T cell enrichment and positive selection using magnetic beads for isolation of CD25⁺ cells. CD4⁺ T cell purity routinely was over 95% and CD25⁺ purity was over 98% with a combined purity of CD4⁺CD25⁺ cells of 90–92% (data not shown). The higher proportion of CD25⁺ versus CD4⁺ T cells suggest that CD4^low/neg CD25⁺ T cells may be positively selected during the isolation procedure. After 3 days of anti-CD3 mAb exposure and an additional 5–8 days in IL-2 alone, cells were subjected to FACS analysis. As shown in Figure 1A, there was an enrichment of a CD4^low/negCD25^int T cell population up to 60% of the final culture. When separated from CD4⁺CD25⁺ T cells and tested in an in vitro suppression assay, CD4^low/negCD25^int T cells were more potent than CD4⁺CD25⁺ T cells in suppressing freshly isolated allogenic stimulated CD4⁺CD25⁻ effector T cells in a dose-dependent manner (Figure 1B).

Figure 1.

Enrichment of a CD4^low/negCD25^int T cell population with potent suppressor function in ex vivo activated and expanded Treg cultures. A, FACS analysis of bead isolated expanded Treg cultures. CD4 surface expression on total Treg cultures and CD25 surface expression on gated CD4⁺ and CD4^low/neg T cells on day 8 of culture are shown. B, Suppressor function of expanded CD4⁺CD25⁺ and CD4^low/negCD25^int T cells. CD4⁺CD25⁺ and CD4^low/negCD25^int T cells were separated by MACS after 8 days of culture and co-cultured with 10⁵ per well CD4⁺CD25⁻ T cells and 10⁵ per well irradiated allogeneic splenic stimulators at the indicated ratios. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from one of five similar experiments is shown (p > 0.01).

To determine whether CD4^low/negCD25^int T cells are derived from CD4⁺CD25⁺ T cells we highly purified freshly isolated Tregs into CD4⁺CD25⁺ T cells and CD4^low/negCD25^int T cells by FACS sorting (purity of both populations > 99%) and then expanded both populations ex vivo in separate cultures (Figure 2A). As compared to cultures containing both cell populations, CD4^low/negCD25^int T cells expanded 17-fold after 11 days of culture in the absence of CD4⁺CD25⁺ T cells (not shown). Importantly, CD4⁺CD25⁺ T cells did not give rise to CD4^low/negCD25^int T cells during culture (Figure 2B). The same result was seen when sorted CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells were reconstituted to the original ratio and identified by FACS based on a congenic marker at the end of the culture (data not shown). In addition CD4^low/negCD25^int T cells derived in the absence of CD4⁺CD25⁺ T cells were highly and comparably suppressive as CD4^low/negCD25^int T cells expanded in the presence of CD4⁺CD25⁺ T cells (Figure 2C). These results suggested that CD4^low/negCD25^int T cells do not derive from or depend on CD4⁺CD25⁺ T cells during the culture. As compared to cultures containing CD4⁺CD25⁺ T cells, we observed that CD4^low/negCD25^int T cells expanded in the absence of CD4⁺CD25⁺ T cells had lower CD4 surface expression levels than when cultured in the presence of CD4⁺CD25⁺ T cells (Figure 2D). We hypothesized that CD4^low/negCD25^int T cells acquired CD4 molecules from CD4⁺CD25⁺ T cells and display it on the surface. In support of this hypothesis, CD4^low/negCD25^int T cells did not express CD4 message as determined by quantitative RT-PCR (data not shown). To exclude the possibility that FACS sorting changed the properties of the sorted cells, CD4⁺CD25⁺ T cells and CD4^low/negCD25^int T cells were sorted and then recombined to the original ratio present before sorting. We did not observe a significant difference in the phenotype (based on CD4 and CD25) and function between non-sorted and reconstituted expanded cultures (Figure 2D and data not shown).

Figure 2.

CD4^low/negCD25^int T cells expand independently from CD4⁺CD25⁺ T cells and acquire suppressor function during in vitro activation and expansion. A, Isolation of highly purified fresh CD4⁺CD25⁺ and CD4^low/negCD25^int T cells. CD4 and CD25 surface expression on freshly isolated Tregs before and after FACS sorting. B, Phenotype of freshly separated and expanded CD4⁺CD25⁺ and CD4^low/negCD25^int T cells. CD4 surface expression of freshly isolated and separately expanded CD4⁺CD25⁺ and CD4^low/negCD25^int T cells on day 11 after culture. C, Suppressor function of freshly separated and expanded CD4⁺CD25⁺ and CD4^low/negCD25^int T cells. CD4⁺CD25⁺ and CD4^low/negCD25^int T cells were co-cultured with 10⁵ per well CD4⁺CD25⁻ T cells and 10⁵ per well irradiated allogeneic splenic stimulators at the indicated ratios. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. D, CD4 surface expression of freshly isolated and expanded cells compared to mixed expanded cell cultures. E, Suppressor function of freshly isolated CD4⁺CD25⁺ and CD4^low/negCD25^int T cells. 2.5×10⁴ freshly isolated CD4⁺CD25⁺ and CD4^low/negCD25^int T cells were co-cultured with CD4⁺CD25⁻ T cells and irradiated allogeneic splenic stimulators. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from one of three similar experiments is shown (C and E).

We next tested the ability of freshly isolated CD4^low/negCD25^int T cells to suppress an alloresponse. Freshly isolated CD4^low/negCD25^int T cells enhanced allo-MLR proliferation (Figure 2E), which required the presence of both responder and stimulator cells (data not shown). Based on these results we conclude that CD4^low/negCD25^int T cell suppressor function is induced during ex vivo activation and culture.

CD4^low/negCD25^int regulatory T cells with suppressive potential are activated, FoxP3⁻ T cells

Because T cells upregulate CD25 surface expression upon stimulation through the TCR the activation status of CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells was determined. Tregs were isolated as in Figure 1 and CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells were analyzed separately based on differential CD4 expression for activation antigen expression by FACS. As determined by size and activation antigen expression (CD62L, CD69, CD44, CD45RB), a higher percentage of freshly CD4^low/negCD25^int T cells were activated as compared to CD4⁺CD25⁺ T cells (Figure 3A). FoxP3 expression was undetectable in CD4^low/negCD25^int T cells. Similarly, when Tregs were activated and expanded ex vivo and CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells analyzed separately based on their differential CD4 expression by FACS CD4^low/negCD25^int T cells maintained an activated phenotype whereas CD4⁺CD25⁺ T cells largely maintained a naïve phenotype. FoxP3 expression was not induced in CD4^low/negCD25^int T cells during the culture period (Figure 3B). When gated on CD4⁺CD25⁺ T cells after expansion only about 30 % of this cell population was FoxP3⁺. In contrast about 80–85% of CD4⁺CD25⁺ T cells in the starting Treg culture were FoxP3⁺ (Figure 3A). Intracellular cytokine expression can be used to further determine the activation status as well as the type of T cell generated upon activation. Expanded Treg cultures were restimulated with pb anti-CD3 mAb in vitro and intracellular cytokine staining was determined in CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells separately analyzed based on the differential expression of CD4. As shown in Figure 3C upon restimulation CD4^low/negCD25^int T cells expressed Th1 cytokines including IL-2, IFN-γ and TNF-α. Even under the activation and expansion conditions, neither IL-4 nor IL-10 was detected. Interestingly, some CD4⁺CD25⁺ T cells also expressed IL-2, IFN-γ and TNF-α but not IL-4 and IL-10 (Figure 3C).

Figure 3.

Characterization of CD4^low/negCD25^int T cells. A, FACS analysis of freshly isolated Tregs gated on CD4⁺CD25⁺ and CD4^low/neg CD25^int T cells. B, FACS analysis of expanded Treg cultures gated on CD4⁺CD25⁺ and CD4^low/neg CD25^int T cells. C, Intracellular cytokine staining of CD4⁺CD25⁺ and CD4^low/neg CD25^int T cells. Expanded Treg cultures were activated with plate-bound anti-CD3 mAb for 4 hours in the presence of 100 U/ml IL-2 and monensin. Cytokine expression gated on CD4⁺CD25⁺ and CD4^low/neg CD25^int T cells is shown. D, FACS analysis of Treg cultures gated on CD4^low/neg CD25^int T cells (dot plots), CD4⁺CD25⁺ and CD4^low/neg CD25^int T cells (CD3 histogram) or CD4⁺CD25⁺, CD4^low/neg CD25^intγδ⁺ and CD4^low/neg CD25^intγδ⁻ T cells (TCR β histogram). Representative plots of at least three experiments are shown.

Several types of naturally occurring and induced T cells with regulatory potential have been described (16). To further characterize the CD4^low/negCD25^int T cells Abs against a panel of cell surface antigens were used in FACS analysis. As shown in Figure 3D all subpopulations were positive for CD3 epsilon and expressed either an αβ or γδ TCR. Some αβ cells co-expressed CD8 or NK1.1 and a subpopulation of γδ T cells also co-expressed NK1.1. γδ⁺NK1.1⁺ T cells were DX5^neg (data not shown). Importantly, expression of activation markers and cytokines and suppressor function was similar in individual subpopulations of CD4^low/negCD25^int T cells (data now shown).

Suppression by CD4^lowCD25^int regulatory T cells occurs independently of IL-10 and TGF-β and requires close proximity to effector T cells and APCs

Some regulatory T cells require IL-10 and TGF-β for their suppressor function, whereas others are thought to suppress independently of these cytokines. To address the requirement for IL-10 and TGF-β in suppressor cell function in CD4^low/negCD25^int T cells Tregs from B6 or IL-10^−/− mice were isolated and expanded ex vivo. CD4^low/negCD25^int T cells from B6 or B6.IL-10^−/− cultures were separated from CD4⁺CD25⁺ T cells at the end of the culture using MACS and were used in a suppression assay. To block TGF-β signaling an anti-TGF-β neutralizing mAb was added to the IL-10 deficient CD4^low/negCD25^int T cells during the suppression assay. Figure 4A shows that IL-10 and TGF-β were not required for suppressor function of CD4^low/negCD25^int T cells in vitro.

Figure 4.

Suppressor mechanisms. A, CD4^low/negCD25^int T cells or CD4⁺CD25⁺ T cells from wt or IL-10^−/− mice were separated after expansion and were co-cultured with 10⁵ per well CD4⁺CD25⁻ T cells and 10⁵ per well irradiated allogeneic splenic stimulators at the indicated ratios in the absence or presence of 100 µg/ml anti-TGF-β mAb. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from one of three similar experiments is shown. B, CD4^low/negCD25^int T cells or CD4⁺CD25⁺ T cells were separated after expansion and were co-cultured with CD4⁺CD25⁻ T cells and irradiated allogeneic splenic stimulators in direct contact or separated by a semi-permeable membrane. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from one of three similar experiments is shown.

Naturally occurring Tregs are thought to suppress in a contact-dependent manner or require close proximity to effector T cells and/or APCs for suppressor function. To test if CD4^low/negCD25^int T cells suppress effector T cells in a similar fashion, CD4^low/negCD25^int T cells were separated from CD4⁺CD25⁺ T cells based on CD4 expression at the end of the culture using MACS and were used in a suppression assay separated from responder T cells and APCs by a semi-permeable membrane. These results showed that contact or close proximity between CD4^low/negCD25^int T cells and responder T cells and APCs was required for their suppressive effect (Figure 4B).

CD4^low/negCD25^int regulatory T cells express cell surface antigens implicated in regulatory T cell phenotype and function

Several surface and effector molecules have been shown to be involved in suppressor function of Tregs, including LAG-3 and CD103 (17, 18, 19). To determine if CD4^low/negCD25^int T cells express some of these suppressor molecules we expanded Tregs ex vivo and analyzed CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells separately based on their differential CD4 expression. The majority of CD4^low/negCD25^int T cells expressed LAG-3 on their surface and a subpopulation expressed CD103 (Figure 5A). In contrast, neither LAG-3 nor CD103 were detected on expanded CD4⁺CD25⁺ T cells. Expression of ICOS and intracellular CTLA-4 was comparable on CD4^low/negCD25^int T cells and CD4⁺CD25⁺ T cells. Expression of the TNFR family members OX40 and glucocorticoid-induced TNFR-related protein (GITR) was higher on CD4⁺CD25⁺ T cells compared to CD4^low/negCD25^int T cells. CD4^low/negCD25^int T cells expressed slightly higher levels of intracellular granzyme B (granB), consistent with their cytokine profile. Expression of molecules shown in Figure 5A did not correlate with any subpopulation of T cells shown in Figure 3D and expression levels were similar.

Figure 5.

Expression and role of Treg molecules in CD4^low/negCD25^int T cells. A, FACS analysis of expanded Treg cultures gated on CD4⁺CD25⁺ and CD4^low/neg CD25^int T cells. B, CD4⁺CD25⁺ T cells, CD4^low/negCD25^int LAG-3⁺ and CD4^low/negCD25^int LAG-3⁻ T cells were sorted by FACS after expansion and were co-cultured with 10⁵ per well CD4⁺CD25⁻ T cells and 10⁵ per well irradiated allogeneic splenic stimulators at the indicated ratios. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from one experiment is shown. C, CD4^low/negCD25^int T cells or CD4⁺CD25⁺ T cells from wt or LAG-3^−/− mice were separated after expansion and were co-cultured with 10⁵ per well CD4⁺CD25⁻ T cells and 10⁵ per well irradiated allogeneic splenic stimulators at the indicated ratios. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from one of two identical experiments is shown. D, CD4⁺CD25⁺ T cells, CD4^low/negCD25^int CD103⁺ and CD4^low/negCD25^int CD103⁻ T cells were sorted by FACS after expansion and were co-cultured with 10⁵ per well CD4⁺CD25⁻ T cells and 10⁵ per well irradiated allogeneic splenic stimulators at the indicated ratios. Proliferation was determined by adding ³H-thymidine at day 6 and cells were harvested 16–18 h later. Average + SEM of triplicate samples from two similar experiments is shown.

To test if LAG-3 is required for the suppressor function in CD4^low/negCD25^int T cells, we sorted expanded cultures into CD4⁺CD25⁺, CD4^low/negCD25^int LAG-3⁺ and CD4^low/negCD25^intLAG-3⁻ populations using FACS sorting. As shown in Figure 5B, CD4^low/negCD25^int that do not express LAG-3 were equally potent as their LAG-3⁺ counterparts in an in vitro suppression assay. To further test if LAG-3 is involved in CD4^low/negCD25^int T cell development, activation or expansion we isolated Tregs from B6 and LAG-3^−/− mice and activated and expanded these cells ex vivo. At the end of culture CD4^low/negCD25^int T cells were separated from CD4⁺CD25⁺ T cells based on CD4 expression by MACS and used in an in vitro suppression assay. No significant difference was observed in the expansion rate between B6 and LAG-3^−/− cultures (data not shown). As shown in Figure 5C we did not observe a significant difference in CD4^low/negCD25^int T cell suppression isolated from wt or LAG-3^−/− mice. To test if suppressor function correlated with CD103 expression we sorted expanded cultures into CD4⁺CD25⁺, CD4^low/negCD25^int CD103⁺ and CD4^low/negCD25^intCD103⁻ populations using FACS sorting. Interestingly, only CD4^low/negCD25^intCD103⁻ T cells were able to suppress an alloresponse very potently (Figure 5D).

Discussion

We show that CD4^low/negCD25^int T cells preexisting at low frequency could outgrow conventional T cells and acquire potent suppressor function, independent of the presence of CD4⁺CD25⁺ T cells, during ex vivo culture. Even though CD4^low/negCD25^int T cells appeared to express low levels of CD4 by FACS quantitative PCR indicated that these cells did not express mRNA for CD4, suggesting passive acquisition of low levels of CD4 antigen from CD4⁺ cells during culture. CD4^low/negCD25^int T cell suppressor function is induced during ex vivo activation and culture, although it remains possible that there is an outgrowth of a highly potent suppressor cell population underrepresented in the initial CD4^low/negCD25^int T cell population that accounts for the suppressor function seen after ex vivo culture. CD4^low/negCD25^int T cells showed an activated phenotype and consisted of several distinct populations, including γδ, CD8 and NKT cells, each of which had similar cell surface antigen expression and cytokine production as well as suppressor cell function. Suppression by CD4^low/negCD25^int T cells required close proximity to effector T cells and APCs as transwells precluded inhibition of effector T cell proliferation and suppression by CD4^low/negCD25^int T cells was not dependent upon the effects of IL-10 or TGF-β regulatory cytokines. Even though a subset of CD4^low/negCD25^int T cells expressed CD103 and the vast majority expressed LAG-3, neither was not required for their suppressor function.

Because freshly isolated Tregs are present at low frequency, ex vivo expansion of Tregs often will be required for therapeutic use in patients and for more detailed characterization of Treg suppression mechanisms. Although several methods for Treg isolation and expansion have been reported, common problems that can hinder these uses include Treg purity and maintenance of potent suppressor function at the end of ex vivo culture. Since T cells other than naturally occurring CD4⁺CD25⁺ Tregs can acquire regulatory properties in vitro or in vivo (16), the finding that suppressor cell function is observed in cultures initiated with a high proportion of naturally occurring Tregs and designed to polyclonally expand T cells does not ensure that suppression is only due to CD4⁺CD25⁺ T cells as we have demonstrated. For example, the reduction in GVHD lethality capacity of polyclonally expanded alloreactive CD4⁺ or CD4⁺CD25⁻ T cells (20) may be due to the presence of other T cell populations such as those reported here. Others have shown that activated CD4⁺ T cells maintained in media supplemented with IL-2 in vitro can be rendered unresponsive to Ag restimulation and can suppress naïve T cell (21). Similarly, in our studies other activated T cell population can acquire suppressor function during in vitro activation and expansion in the presence of exogenous IL-2.

Polyclonal Treg expansion conditions often are utilized for preclinical studies and will be incorporated into many therapeutic trials especially in instances in which there are numerous or unknown target peptides that can be loaded onto APCs used to drive antigen-specific Treg expansion or there are undesirable effects of using host APCs as might be in instance in which the patient has a hematopoietic malignancy. Therefore the need to develop expansion of polyclonal Tregs, as used here, has been a major focus of the field. For clinical applications, our data raise the question as to whether it always is advantageous to initiate cultures with a highly purified population if bystander cells could contribute to suppression either directly or via support Treg expansion. Further, our data highlight the need for detailed characterization of ex vivo activated and expanded Treg cells cultures in rodent and human systems. The use of ex vivo expanded cells for studies designed to characterize Tregs may be compromised by the reliance exclusively upon suppressor cell function as the major read-out of preservation of Treg function at the end of culture since other non-Treg populations clearly can dominate the suppressor cell assay response. Limited flow cytometry analysis of ex vivo expanded Treg containing populations may be insufficient especially considering that murine Tregs can lose FoxP3 during culture or FoxP3⁻ cells can outgrow ex vivo cultures, whereas activated human CD4⁺CD25⁻ T cells can acquire FoxP3 expression. The use of highly purified, cell-sorter isolated FoxP3-GFP transgenic CD4⁺CD25^hi cells could be used to avoid such complications of expansion, although this method cannot be applied to human Tregs. Drugs such as rapamycin that favor the development of suppressor cell function warrant investigation in cultures such as these to determine the extent to which the increased potency of suppression is due to effects on pre-formed Tregs.

Although several groups have identified NK1.1⁺ γδ T cells, this is the first report to show that this population can be propagated from ex vivo expanded Treg cultures (22–25). Drobyski et al., showed that ex vivo activated γδ T cells could delay GVHD when administered 2 weeks prior to effector T cell infusion suggesting a regulatory function of expanded γδ T cells in vivo (26). Double-negative (DN) TCRαβ regulatory T cells have been identified in rodents and humans (27). A recently published report showed that DN regulatory T cells could be derived from Tregs expanded ex vivo and in vivo (28). These Treg could be derived DN-regulatory T cells lost FoxP3 expression and their suppressor function was dependent on perforin. In mice, such cells, present at very low numbers in peripheral lymphoid tissues, can be expanded in vitro and upon adoptive transfer into irradiated allogeneic recipients, will prevent graft rejection and GVHD. In our study we generated activated T cells with suppressor function that were independent of Tregs. Together these studies highlight the complexity of regulatory T cell phenotypes as well as mechanisms of suppressor functions of suppressor T cells expanded ex vivo.

The suppressor mechanisms of naturally occurring Tregs are still largely unknown. In vitro suppression is thought to be contact-dependent or rather dependent on close proximity to effector T cells and APCs. Most induced regulatory T cells secrete IL-10 and TGF-β or secrete Th2 type cytokines (16). The CD4^low/negCD25^int T cells described in this study produce a large amount of Th1 type cytokines involved in cytotoxic immune responses. Similar to our findings with CD4^low/negCD25^int T cells, neither IL-10 and TGF-β (29) are required for in vitro suppression by naturally occurring Tregs, although this is not uniformly the case for Tregs in vivo (30–32). CD4^low/negCD25^int T cells could limit alloresponses by targeting alloreactive T cells or allo-APCs directly by releasing these cytokines or through granzyme B and perforin (33, 34). Although blocking LAG-3 has been shown to abrogate suppressor function in natural Tregs and Ag-specific transgenic Tregs (17) and even though a large fraction of CD4^low/negCD25^int T cells were LAG-3⁺, LAG-3 expression was not required for their suppressor function. Our data also showed that CD4^low/negCD25^intCD103⁺ T cells did not suppress as well as their CD103⁻ counterpart. CD103 is a recently identified marker for a subset of Tregs that is important for immune regulation in vivo. Because CD103 does not seem to have suppressor function itself but rather is important in Treg homing or retention (19, 35), it is likely that the expression of CD103 demarcates a subpopulation of CD4^low/negCD25^int T cells and itself does not influence suppressor cell function.

In conclusion, we report the identification and characterization of CD4^low/negCD25^int T cell population that consisted of subpopulations of T cells expressing either an αβ or γδ T cell receptor. Such cells had an activated phenotype, did not express the transcription factor FoxP3, and despite the expression of LAG-3 and CD103, suppressor cell function was not dependent upon LAG-3 or CD103 expression. Because CD4^low/negCD25^int T cells were more than 20 fold more potent than expanded conventional Tregs in suppressing an in vitro CD4⁺CD25⁻ T cell response to allo-antigen, ex vivo expansion cultures should be carefully monitored by flow cytometry for the existence of non-Treg populations and further indicate that suppression potency can be striking augmented by non-Treg populations present at low frequency at the time of culture initiation.

Abbreviations used in this paper

GITR

glucocorticoid-induced TNFR-related protein

LAG-3

lymphocyte activation gene-3

Treg

CD4⁺CD25⁺ regulatory T cells

pb

plate-bound

DN

double-negative