In vitro–expanded human CD4⁺CD25⁺ T-regulatory cells can markedly inhibit allogeneic dendritic cell–stimulated MLR cultures

It is becoming increasingly clear that naturally arising CD4⁺CD25⁺ T regulatory (Treg) cells can influence most immune responses.^1,2  Initially they were described to be critical for the control of autoimmunity,^3-5  and more recently, they have been shown to regulate transplantation tolerance,^6,7  antitumor immunity,^8,9  and anti-infectious responses.^10,11  Thus, these cells appear to be central control elements of immunoregulation, and understanding their biology will be important for efforts aimed at therapeutically manipulating immune responses.

Treg cells were first characterized in mice, where they constitute 6% to 10% of lymph node and splenic CD4⁺ T-cell populations. They are generated both through central thymic developmental mechanisms in pathogen-free mice¹²  and also arise by poorly defined peripheral generation or expansion mechanisms.^13-15  It has been demonstrated that after antigen-specific activation, Treg cells can nonspecifically suppress proliferation of either CD4⁺ or CD8⁺ T cells.^15,16  The mechanism of suppression is unclear and, in vitro, appears to require cell-cell contact. A significant subsequent result of suppression is impaired production of interleukin 2 (IL-2).^15-17  In vivo, the suppression mechanism is more controversial, with some studies showing dependence on immunosuppressive cytokines,^15,18  which are not required for in vitro suppression.

In tissue culture, CD4⁺CD25⁺ cells are naturally anergic and hyporesponsive even with strong antibody-based stimulation.^16,17  However, with the provision of exogenous IL-2, they can proliferate to antigen-loaded antigen-presenting cells (APCs) or to anti-CD3-based stimulation.^16,17  Initial studies revealed that anti-CD3 plus IL-2-based culture enabled limited expansion of functional murine Treg cells over the course of a 7-day culture.^19,20  On a per cell basis these cells were 4- to 6-fold more potent at suppression.¹⁹  Later work has demonstrated that multiple restimulations of murine Treg cells can lead to loss of function.²¹  Studies in mouse models of bone marrow transplantation (BMT) have shown that CD4⁺CD25⁺ cells can prevent graft-versus-host disease (GVHD) across major histocompatibility complex barriers.^22-24  Our previous studies have shown that ex vivo polyclonally expanded Treg cells, with anti-CD3 plus IL-2 (for 10 days), can be effective in preventing GVHD.²²  Studies by others have shown that ex vivo expansion of Treg cells with irradiated allogeneic APCs plus exogenous IL-2 can suppress GVHD.^24,25  Subsequent studies have shown that Treg cells can prevent GVHD and still allow for antitumor or graft-versus-leukemia (GVL) effects.^25-27  Thus, Treg cells could have a potential role in clinical immunosuppressive therapy in transplantation, provided human Treg cells could be isolated and expanded in culture to provide sufficient numbers for in vivo infusion.

Studies of human CD4⁺CD25⁺ Treg cells have lagged behind those of murine cells but are becoming increasingly reported. These cells are present in smaller numbers in human peripheral blood than in murine lymph node or spleen, and in addition, the population of CD25⁺ cells is less distinct in human blood because there is an overlapping population of CD25^dim cells. It has been reported that only the brightest 1% to 2% of CD25 expressers, which after isolation by fluorescence-activated cell sorting (FACS), have suppressor function (in low-dose immobilized anti-CD3 monoclonal antibody [mAb]-based coculture suppressor assays).²⁸  The potency of suppression exhibited by freshly isolated human CD4⁺CD25⁺ cells has been modest in phytohemagglutinin or soluble anti-CD3 mAb-based assays.^29-31  The best suppressor activity has been obtained by 2 groups who used dendritic cell (DC)-stimulated mixed lymphocyte reactions (MLRs) for assay.^32,33  In the only report of human CD4⁺CD25⁺ polyclonal expansion, using soluble anti-CD3, and lymphoblastoid cells and peripheral blood mononuclear cells (PBMCs) as feeder cells, plus IL-2, the suppressive function noted (a 65% reduction of proliferation at a suppressor-responder cell ratio of 1:1) was less than typically observed with mouse Treg cells.³⁴  Short-term antigen-specific cultures have been reported using DCs plus cytokine supplementation, but these lines have generally had limited expansion capability even after several rounds of stimulation.³⁵ 

To better characterize CD4⁺CD25⁺ Treg cell function, we sought to develop improved means to isolate and culture these important cells. Here we show that stringently purified, polyclonally activated (with anti-CD3/28 mAb-coated beads and CD4⁺ feeder cells), human CD4⁺CD25⁺ cells can form cell lines that exhibit enhanced suppressor function. Hence, we characterized the phenotype and function of these culture-expanded potent Treg cells. These cell lines exhibit a profound suppressive bioactivity and form a model system for the study of Treg cell biology.

T cells were isolated from buffy coat preparations derived from the whole blood of healthy volunteer donors (Memorial Blood Centers, Minneapolis, MN). Leukocyte-rich buffy coat cells were centrifuged over Ficoll-Hypaque layers to collect PBMCs. CD25⁺ cells were isolated by positive selection from PBMCs with directly conjugated anti-CD25 magnetic microbeads (2 μL/10⁷ cells; Miltenyi Biotec, Auburn, CA), and purified over an LS⁺ column. Cells were then applied to a second magnetic column, washed, and eluted again. After the double-column procedure, cells were routinely more than 93% pure (for CD25) by FACS analysis. Alternatively, cells were indirectly stained with anti-CD25-fluorescein isothiocyanate (FITC), clone 2A3 (Becton Dickinson Immunocytometry Systems, San Jose, CA), washed, and bound to anti-FITC multisort microbeads (3 μL/10⁷ cells; Miltenyi Biotec) and positively selected. As with the direct microbead system, cells were reapplied to a second column. After column purification, multisort beads were detached, and the CD25⁺ cells were depleted of cells expressing CD8, CD14, CD19, CD20, and CD56 with a cocktail of mAb-coated microbeads for lineage depletion.

The non-CD25 fraction of PBMCs was further depleted of CD25⁺ cells with more anti-CD25 microbeads (10 μL/10⁷ cells). CD4⁺ T cells were then isolated from the CD25 fraction by positive selection with anti-CD4 mAb-coated magnetic microbeads (10 μL/10⁷ cells; Miltenyi Biotec). Cells were routinely 96% to 98% pure CD4⁺CD25^- by FACS analysis.

Isolated CD4⁺CD25⁺ cells or control CD4⁺CD25^- cells were cultured with anti-CD3/CD28 mAb-coated Dynabeads (University of Pennsylvania, Philadelphia)^36,37  at a 2:1 bead-total cell ratio. CD4⁺CD25^- feeder cells were irradiated at 30 Gy and added at a 1:1 ratio to CD4⁺CD25⁺ cells. Cells were cultured at 1 million (nonirradiated) cells/mL in 24-well plates. IL-2 was added on day 3 at 50 IU/mL (Chiron, Emeryville, CA). Cells were split as needed, approximately one third every 3 days during the fast-growth phase. Culture media was RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Gibco), l-glutamine, penicillin, and streptomycin.

Immature DCs were generated from CD14⁺ monocytes,^38,39  isolated from PBMCs, by magnetic bead-based purification (Miltenyi Biotec), and were cultured in X-Vivo-15 (BioWhittaker, Walkersville, MD) media at 1 million cells/mL supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/mL final) and IL-4 (20 ng/mL final) cytokines from R&D Systems (Minneapolis, MN). Cells were cultured for 5 to 10 days before use as stimulators in MLRs. For some experiments, DCs were matured with tumor necrosis factor α (TNF-α; 20 ng/mL final) and poly (I:C), a Toll-like receptor 3 (TLR-3) agonist ligand (20 μg/mL final; Sigma, St Louis, MO) for 2 days.^40-42  In other experiments, the TNF and Poly (I:C) (at the same concentrations), or lipopolysaccharide (LPS; 10-1000 ng/mL; Sigma) were added directly to MLRs. DC stimulators were irradiated at 30 Gy.

A total of 5 × 10⁴ responding CD4⁺CD25^- T cells and 5 × 10³ DC stimulator APCs were cultured per well in 96-well U-bottom plates. Test cultured suppressor or conventional T-cell lines were added at 2.5 × 10⁴/well for standard assays or in graded numbers for titration experiments. For antibody-blocking experiments, 1 × 10⁴ suppressor cells were used. Culture media was RPMI 1640 (Gibco) supplemented with 10% FCS (Gibco), l-glutamine, penicillin, and streptomycin. Wells were pulsed on days 3, 5, 6, and 7 with ³H-thymidine for the last 16 hours of culture. All time points had 6 replicates. Results are expressed in counts per minute. Data are collected with direct beta counter; hence, our reported counts per minute are lower than typically reported with liquid scintillation amplification. Thus, the absolute magnitude of counts is lower, but the comparative differences between experimental samples remain the same.

Culture supernatants were spun free of cells and aliquots were frozen at -80°C. Supernatants were evaluated by the Luminex assay system with a latex bead-based multianalyte system (R&D Systems).

Cultured suppressor cell lines were tested for cytotoxicity against allogeneic DCs or the natural killer (NK) cell-sensitive cell line K562 in 4-hour ⁵¹Cr-release assays. Effector-to-target ratios ranged from 20:1 to 0.6:1. Target cells were labeled with sodium chromate-⁵¹Cr (DuPont, Wilmington, DE) for 60 minutes. All determinations were performed in triplicate and the percentage of lysis was determined. Positive control lytic NK92 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in the presence of 500 U/mL recombinant human IL-2 (Chiron).

To follow purification, cells were stained with an anti-CD25-phycoerythrin (PE), which is not blocked by anti-CD25 microbeads, clone M-A251 (BD Pharmingen, San Diego, CA). Other antibodies for flow cytometry included anti-CD4-PerCP (clone SK3), anti-CD8-PerCP (SK1), and anti-CD19-APC (4G7) from Becton Dickinson Immunocytometry Systems; anti-CD27-FITC (M-T271), anti-CD62L-PE (Dreg 56), anti-CD69-FITC (FN50), anti-CD152-PE (BNI3), anti-CD122-PE (Mik-b2), anti-CD132-PE (AG184), and anti-CD134 (ACT35) from BD Pharmingen; and anti-CCR7-PE (no. 150503) and anti-glucocorticoid-induced TNF receptor (anti-GITR)-PE (no. 110416) from R&D Systems. In functional experiments aimed at blocking suppression, neutralizing antibodies were used at titered amounts up to 20 μg/mL. Antibodies included anti-IL-10 (23738), anti-IL10-receptor-α (37607), and anti-transforming growth factor β-1, -2, -3 (anti-TGF-β-1,-2,-3) (1D11) from R&D Systems.

For immunofluorescence staining, cells were stained for 30 minutes at 4°C, with titered amount of each antibody. Cells were washed again and analyzed on a FACS Calibur cytometer (BD Immunocytometry Systems). Cell line subsets were sorted on a FACS Vantage. Data were analyzed by FloJo software version 4.4 (Treestar, Ashland, OR). Intracellular staining was done using paraformaldehyde-fixed cells, 2% at room temperature for 30 minutes, followed by permeabilization and staining for 1 hour, and washing in 0.1% saponin-containing buffer, phosphate-buffered saline (PBS) plus 5% FCS-5% human AB serum.

All error bars represent 1 SD above and below the mean. A paired, 2-tailed Student t test was used to determine the statistical significance of differences between proliferative responses. P values less than .05 were considered significant.

Human peripheral blood contains a small population of cells that express the Treg phenotype, positive for both CD4 and CD25 antigens (and distinctly positive for CD25; Figure 1A). This population represents approximately 1% to 2% of PBMCs, or 3% to 5% of CD4⁺ T cells (mean, 3.8%; range, 2.4%-6.3% of CD4⁺ T cells; n = 20). Some donors exhibit a more distinct population of CD4⁺ and CD25⁺ cells (Figure 1B). However, there exists a large population of partially overlapping CD25^dim cells, which necessitates stringent purification for the isolation of the CD25⁺ Treg cells. To more specifically isolate the CD25⁺ Treg cells we developed a modified magnetic-activated cell sorting (MACS) purification. Using a lower titer of anti-CD25 mAb-coated microbeads led to isolation of cells with higher mean channel fluorescent intensity of CD25 expression. In addition, reapplication of the magnetically isolated cells to a second column for additional purification further increased the enrichment of CD25⁺ cells. The use of cleavable microbeads allowed for removal of beads after CD25 purification, which then permitted subsequent lineage depletion (of CD8, CD14, CD19, and CD56 cells). This strategy led to the generation of highly purified CD4⁺CD25⁺ cell populations (Figure 1C). The CD25^- fraction of cells was further depleted of CD25⁺ cells and used as the source for purification of CD4⁺CD25^- cells, for isolation of conventional T-cell controls (Figure 1D).

Human CD4⁺CD25⁺ Treg cells are hyporesponsive to stimulation with anti-CD3 mAb or DCs. However, they can proliferate when given these stimuli plus IL-2 or IL-15, albeit to a much lower extent than CD4⁺CD25^- T cells.^32,33  For our initial experiments, purified CD4⁺CD25⁺ cells were expanded with immobilized anti-CD3 plus IL-2, which allowed for a 5- to 10-fold expansion over 2 weeks. To further augment expansion potential, a costimulatory molecule-based stimulation was investigated. To do this, we used cell-sized Dynabeads with anti-CD3 and anti-CD28 mAbs covalently attached (designated 3/28 beads). This reagent has been used successfully for clinical-scale expansion of conventional T cells for immunotherapy trials,^36,37  and has enabled more than one million-fold expansion of T cells. Quite dramatically, however, we found that the stringently purified CD25⁺ Treg cells proliferated poorly with stimulation with the 3/28 beads alone. This contrasts with the vigorous response generated by the CD4⁺CD25^- cells (Figure 2A). The weak response of CD4⁺CD25⁺ cells is significantly augmented by IL-2 supplementation, and this combination is sufficient for modest Treg expansion from most donors (Figure 2B). However, the more stringently the CD4⁺CD25⁺ cells were purified, the less well they grew in culture, even with stimulation with 3/28 beads and IL-2.

Because CD4⁺CD25⁺ T cells appear to have defects in cytokine production,^32,33  we reasoned that conventional T cells could complement this deficiency and provide for augmented expansion. Irradiated CD4⁺CD25^- feeder cells were added to 3/28 bead-stimulated Treg cultures at initiation (1:1 ratio) and provided for a sustained increase in proliferative response (Figure 2B). This augmentation was significantly greater than that with IL-2 alone at 100 IU/mL. Interestingly, supplementation of Treg cultures with conditioned media (20% vol/vol; derived from activated conventional CD4⁺CD25^- T cells on day 5 after 3/28 bead stimulation) could largely (but not completely) reproduce the effects of feeder cells. This suggests that the activated conventional T cells produce soluble growth factors for CD4⁺CD25⁺ cell expansion (primarily IL-2, but possibly other factors). Importantly, these feeder cell-supplemented cell lines maintained potent suppressor function.

With the use of 3/28 beads, IL-2, and CD4⁺CD25^- feeders, the CD4⁺CD25⁺-derived cell lines exhibited significant growth, and over 100-fold expansion was readily obtainable. The expansion occurred as a classical sigmoid growth curve, beginning slowly, rapidly expanding over 1 to 2 weeks, and then reaching a plateau phase (Figure 2C). After reaching a growth plateau phase, cell lines were maintained in IL-2, and suppressor function was sustained for 3 to 6 weeks. In some cases cell lines in culture for up to 3 months had potent suppressive activity; however, typically suppressor function diminished over time (not shown).

All cell lines were initially screened for suppressor activity in MLRs after 2 to 3 weeks of culture and then further analyzed over the next 3 to 4 weeks. To evaluate suppressor function, we used an HLA-mismatched allo-MLR assay as a functional readout. Purified freshly isolated CD4⁺CD25^- T cells were reacted with irradiated immature DCs from unrelated donors. Test cells (cultured CD4⁺CD25⁺ and CD4⁺CD25^--derived cell lines) were added to the MLR at day 0 with a regulator-responder ratio of 1:2. Suppression was reflected by decreased proliferation and was most evident on day 6 to 7, peak of control MLR. These assays are very robust and consistent among donors and therefore served as our standard measure of suppression.

The majority of cell lines derived from CD4⁺CD25⁺ cells (19 of 25, 76%) had clear suppressive function (> 65% inhibition of proliferation) at a suppressor-responder ratio of 1:2 (Figure 3A). In contrast, cell lines derived from CD4⁺CD25^- cells were found invariantly to augment the MLR (Figure 3A). The remaining CD4⁺CD25⁺-derived cell lines (6 of 25, 24%) had weak suppressive function (20%-65% inhibition of proliferation). However, none augmented the MLR (Figure 3B). Consistent with prior observations,³²  freshly isolated MACS-purified CD4⁺CD25⁺ cells have only modest suppressive activity in these MLR assays, equivalent to the weak suppressor cell lines (20%-65% inhibition of proliferation; Figure 3B). Importantly, 9 (47%) of 19 of the suppressive cell lines had potent suppressor activity, and these cell lines almost completely inhibit MLR cultures (> 90% inhibition of proliferation; Figure 3C). The level of suppressive activity was an intrinsic characteristic of each line, in that weak or potent suppressive cell lines had consistently similar activity when tested in multiple independent MLR experiments over several weeks of analysis (see “Characterization of suppressor cell function in MLR assays”).

Potent suppressor cell lines were cultured in parallel with CD4⁺ CD25^--derived cell lines from the same individual, which served as conventional T-cell controls. The weakly suppressive cell lines were also characterized and compared to determine the distinguishing characteristics versus the potently suppressive cell lines. Interestingly, it was often possible to predict suppressor function based simply on growth characteristics in culture, where the most rapidly growing CD4⁺CD25⁺ lines generally had the least suppressive function (not shown).

All cell lines initially expressed a somewhat typical activated T-cell phenotype after stimulation with the 3/28 beads. The cell lines transiently expressed relatively equivalent amounts of activation antigens, which quickly diminished over the 2 to 3 weeks after activation. These include CD122, CD132, GITR, OX40 (CD134), and cell surface CTLA4 (CD152). However, after several weeks of culture, when the cells become relatively quiescent (maintained in IL-2), the phenotypes become clearly divergent. Compared with T-cell lines derived from CD25^- cells, the strongly suppressive Treg cell lines expressed higher levels of CD25, and the elevated expression was sustained. The levels after 3 weeks of culture (mean fluorescence intensity [MFI] 22 versus 210) are shown (Figure 4A-B). In addition, intracellular staining for CTLA4 demonstrated enhanced expression in the suppressor cell lines (MFI 8 versus 64; Figure 4D-E). The differences in CD25 and intracellular CTLA4 expression were the most distinct phenotypic characteristics identified in our studies that distinguish conventional versus CD25⁺-derived cell lines. The weakly suppressive cell lines expressed intermediate amounts of these 2 key descriptive antigens (Figure 4C,F).

To determine further differences between the weakly and potently suppressive cell lines, additional cell surface antigen analysis was undertaken. We noted a correlation of 3 antigens (CD62L, CCR7, and CD27) that were expressed on a higher percentage of cells in the potent suppressor cell lines compared with weak lines (Figure 4G-I). To further evaluate for functional relevance, we used magnetic beads to isolate CD62L⁺ or CD27⁺ cells (the brightest 2 antigens) and found enrichment for suppressor activity in both of the positive subsets (not shown). To more definitively determine the function of these cell line subsets, cell lines were sorted for CD62L⁺/CD27⁺, CD62L⁺/CD27^-, and CD62L^-/CD27⁺ cells (Figure 4J) and each population was tested for suppressor activity in MLRs. Suppressor function was solely within the CD62L⁺/CD27⁺ subset. In contrast, the other subsets were found to augment the MLR (Figure 4K). Thus, CD4⁺CD25⁺ cell lines can contain mixtures of suppressive and nonsuppressive cells, and the suppressive effects can be dominant over the augmenting effects of the nonsuppressors. CD62L and CD27 coexpression can be used to distinguish these subsets and facilitate selection of cell lines (or cell line subsets) with potent suppressive potential.

To determine the cellular mechanism of suppression in the MLR assays, potent cell lines (> 90% day 6 MLR suppression) were selected for further analysis. They were first shown to be consistently suppressive and then titered to determine minimum number required for potent suppression. To determine how broadly reactive the cultured Treg cells are, several independent lines were tested for suppression in 8 separate HLA-mismatched MLR cultures. In all cases, the cultured Treg cells markedly suppressed all MLR cultures analyzed (Figure 5A). Although there was some variation in the magnitude of suppression (mean, 92%; range, 81%-98%; n = 16), potent Treg cell lines were effective in inhibiting almost all MLR cultures. To quantitate the minimum number of suppressor cells required for potent inhibition, titrated numbers of suppressor cells were added to indicator MLR cultures. The titration curves (Figure 5B) revealed an approximate break point at a suppressor-responder ratio of less than 1:10 (5000 suppressors to 50 000 responders). The titration curves were nonlinear, possibly indicating cooperative effects in the overriding of suppression with low Treg cell dose.

Proliferation was nearly completely impaired in suppressed MLRs at all time points, suggesting that the suppressor effects occur within the first 3 days, that is, prior to the proliferative burst in MLRs. To evaluate for earlier effects and search for potential regulatory cell deviation of the quality of immune response, MLR supernatants were evaluated for cytokine content. There was a profound suppression of cytokine accumulation. Suppressed MLRs make a minimally detectable small early wave of IL-2 (at the threshold of sensitivity of assay), with no late production detectable (Figure 5C). Because the control MLR manifested IL-2 accumulation in the supernatant, even 1 day after initiation of culture, the suppressor effect was detectable and already profound as early as the first day of the suppressed MLR. In addition, accumulation of late cytokines (peaks typically day 5-7), such as TNF-α, interferon γ (IFN-γ), GM-CSF, and IL-6, was nearly completely prevented throughout the culture (Figure 5D). There was no induction of IL-4 or IL-10 to indicate deviation to a T_H2 or T-regulatory type 1 (Tr1/IL-10 producing) differentiation. In fact, IL-10 accumulation is prevented as well.

To examine responder T-cell reactivity early in the MLR, cells were assayed by flow cytometry for activation marker expression. Using responder cells derived from an HLA-A2⁺ donor, and suppressor cells derived from an HLA-A2^- donor, the responding cells could be distinguished during coculture. Control and suppressed MLR cells were evaluated 24 or 48 hours after initiation of culture for induction of CD69, CD25, and OX40 (CD134). In the control MLR, the 2% to 4% of responder T cells showed expression of these activation antigens (Figure 6A-C), consistent with the expected alloreactive T-cell frequency for HLA-mismatched MLRs. Notably, in the suppressed MLRs very few responder T cells showed expression of these activation antigens (Figure 6D-F). These data further suggest a very early block in T-cell activation as a mechanism of Treg cell action.

The potency of suppression was remarkably retained if the DCs stimulating the MLRs were activated or matured. Maturation/activation of DCs with lipopolysaccharide (LPS), a TLR4 ligand, or the combination of TNF/poly (I:C), a double-stranded RNA analog-TLR 3 ligand,^41,42  did not lead to bypass of suppression (Figure 6G). Inclusion of LPS or TNF/poly (I:C) in the MLR culture also did not bypass suppression. Thus, the cultured human Treg cells were very potent, and activated DCs, which express abundant costimulatory molecules and cytokines, were not able to bypass their suppressive effect.

The potent and early inhibition of MLRs suggests APC inactivation or elimination as a possible mechanism of suppression. However, by microscopic evaluation, the DCs appear to persist for the duration of culture. In addition, the suppressor cell lines did not have cytotoxicity for allogeneic DCs (Figure 7A) or NK/LAK-sensitive targets (K562; Figure 7B) in chromium-release assays.

To determine if known immunosuppressive factors were mediating the action of the cultured Treg cells, antibodies capable of neutralizing IL-10 or TGF-β were added to control and suppressed MLR cultures. Because of the potency of the suppressive effect, lower numbers of suppressors were added to make the assay more sensitive to reversal of suppression. Despite this, antibodies reactive with IL-10, IL10R-α, or TGF-β-1, -2, -3 failed to reverse suppression and the inclusion of all 3 antibodies together had a very modest effect (Figure 7C). Doses of 1, 10, or 20 μg/mL were tested with the effects only noted at the highest dose levels. In addition, transwell studies were undertaken to determine if soluble factors released by Treg cells could convey suppression. Indicator MLR cells were cultured above resting or activated Treg cells or suppressed MLR cultures, separated by membranes with 0.4-μm pores. No suppressive effect was found to pass through the membrane (not shown).

Importantly, when cultured Treg cells were added to allo-MLRs driven by APCs that were derived from the same donor as the suppressors, but still allogeneic to responder T cells, minimal suppression was noted (Figure 7D). Thus, when cultured Treg cells derived from donor A are added to an allo-MLR driven by DCs from the same donor A, there is minimal suppression. However, when these same Treg cells are added to an MLR driven by DCs from donor B, there is suppression. These results indicate that the cultured Treg cells are not constitutively suppressive of all MLRs. The cultured Treg cells need some form of specific additional stimulation that can be provided by allo-DCs (and not by autologous DCs).

In this study, we demonstrate that suppressor cells can be isolated and expanded in culture from human blood. Importantly, these cultured Treg cells can be expanded over 100-fold and, when pure, express enhanced and potent suppressive activity. We have characterized the suppressive function of these cells in MLR assays and shown that they can nearly completely block HLA-mismatched MLRs. These culture-expanded suppressor cells have many of the hallmark features of the freshly isolated CD4⁺CD25⁺ Treg cells. They highly express CD25 and cytoplasmic CTLA4, their activity is dependent on cell-cell contact, and suppression does not seem to be dependent on cytolytic activity or on the immunosuppressive cytokines IL-10 or TGF-β.

Prior work has noted the need in the human system of isolating the CD25^+bright subset of CD4⁺CD25⁺ cells to detect suppressor activity (with freshly isolated cells) in antibody-based coculture assays.²⁸  We have found a similar situation in our culture system, where the most stringently purified CD4⁺CD25⁺ cells form the best suppressor cell line precursors. Contaminating CD25^dim cells in CD25⁺ fractions can grow faster and overgrow the CD25^+bright cells, and thereby preclude the full manifestation of suppressor cell function. Thus, we emphasized stringent purification. We found that lower titers of anti-CD25 magnetic microbeads (one fifth of the manufacturer's recommendation) and a repurification over a second column greatly facilitated the generation of Treg cell lines with potent suppressive capabilities. The use of even lower titers of anti-CD25 mAb-coated magnetic micobeads or addition of a third column step to the purification did not significantly improve results and had the disadvantage of decreasing yields.

To our knowledge, these data are the first to demonstrate the feasibility of anti-CD3/28 mAb-coated bead-based approach for Treg cell activation and expansion. When anti-CD28 is combined with anti-CD3 mAb a more efficient expansion strategy results. There was some initial concern that anti-CD28 mAb costimulation may lead to reversal of suppression, as noted in short-term murine suppressor assays.^17,19  Our findings indicate that anti-CD28 mAb-mediated expansion of human Treg cells does not impair suppressor ability directly over the long-term and may actually increase it. In addition, we and others have recently demonstrated that soluble anti-CD28 mAb does not lead to bypass of suppression in anti-CD3 mAb-based suppression assays in the human system (not shown).¹⁴  Interestingly, in the rat, anti-CD28 superagonist mAbs (particular epitope-specific anti-CD28 mAbs alone signal as a mitogen, and not just as a costimulator) alone can preferentially enhance expansion of CD4⁺CD25⁺ cells in vivo.⁴³  Thus, CD28 signals may not only be important for homeostatic expansion of Treg cells but may also be used to overdrive their accumulation. Despite anti-CD3/CD28 mAb-coated bead-based and IL-2-supplemented activation, stringently purified CD4⁺CD25⁺ cells (or FACS sorted CD4⁺CD25^+bright cells) did not grow well. Growth was better with the addition of CD4⁺CD25^- feeders, which provide IL-2 and other growth factors.

CD4⁺CD25⁺ cell line growth and suppressor function were variable and inversely correlated. We favor the interpretation that cell lines with poor suppressor function were the result of inclusion of small numbers of conventional T cells in the CD4⁺CD25⁺ cultures. Cell lines derived from more stringently purified cells did not grow as well but were more potent suppressors. In addition, the immunophenotype of the weakly suppressive cell lines (CD25 moderate and split CD62L or CD27) was consistent with a mixed population of conventional and suppressive cells. Interestingly, after culture, the functionally active suppressor cells were exclusively within the CD62L⁺ and CD27⁺ double-positive population, and the potent suppressor cell lines nearly uniformly expressed these antigens.

In our functional analysis we demonstrated that the potent cultured Treg cells can nearly completely block an allo-MLR. With these cell lines, the marked suppressive effects could occur with as little as 3125 suppressors (1:16 ratio) in a culture of 50 000 responding CD4⁺ T cells and 5000 DC stimulators. These profound effects are more potent than what has previously been reported for freshly isolated^32-34  or expanded human CD4⁺CD25⁺ cells.³⁴  The potent suppressor cell lines prevented accumulation of all the cytokines tested that were produced in control cultures (IL-2, TNF-α, IFN-γ, GM-CSF, IL-6, and IL-10). The block in T-cell activation appeared to occur very early because a marked suppressive effect on activation antigen expression and IL-2 accumulation was clearly evident on the first day of culture. This suggests the hypothesis that Treg function in MLR cultures predominantly reflects impairment of T-cell activation.

Surprisingly, the activation or maturation of the DCs did not lead to the bypass of suppression. This finding is in contrast to what has been recently reported for freshly isolated murine Treg cells, where LPS or CpG-containing DNA oligodeoxynucleotide-mediated signaling of spleen-derived DCs led to the bypass of suppression.⁴⁴  However, in that system the Treg cells were not culture activated and expanded. In this system, the activated and expanded human Treg cells can override the cytokines and costimulatory molecules expressed by activated DCs and still block the MLR response. The finding of increased potency of suppressor function after culture is consistent with what has been shown with activation of murine Treg cells. Suppressor function is activation dependent,¹⁶  and short-term culture with anti-CD3 and IL-2 augments suppressive ability.²⁰  We favor the interpretation that the cultured Treg cells are primed (more sensitive) to reactivation of T-cell receptor (TCR), and hence TCR-induced suppressor function is more readily expressed. IL-2 (in conjunction with TCR signaling) has also recently been shown to be important for up-regulation of suppressor function.⁴⁵ 

Murine studies have revealed that under a number of conditions tested, freshly isolated or cultured Treg cells have been able to prevent GVHD and still allow for GVL activity,^25-27  or even immune reconstitution.²⁵  It has been suggested that Treg cells can inhibit alloreactive T-cell expansion and cytokine production (more important for GVHD induction) more so than CTL activity (more important for antitumor activity), and some data supporting this model have been presented.²⁷  If these concepts and mechanisms accurately reflect human disease pathogenesis, then the provision of cultured Tregs to patients may provide for improved outcomes in BMT.

The availability of large numbers of cultured Treg cells will enable more detailed immunologic, biochemical, and molecular characterization of these important cells. In addition, because our procedure is adaptable for good manufacturing practice (GMP) conditions, clinical testing may soon be feasible, and cultured Treg cells may be useful as a novel form of immunosuppressive therapy.