nTreg cells out-compete naïve CD4+T cells in the formation of DC aggregates
It has previously been demonstrated in vitro that CD4+CD25+ nTreg cells can out-compete naïve T cells in aggregating around splenic DCs in an assay using the OVA-specific DO11.10 TCR transgenic system (H-2d) []. This competitive ability of nTreg cells was considered a potential mechanism of suppression of naïve T-cell activation. Here, we asked whether TGF-β-induced Treg cells could function in a similar manner and, in addition, whether these Treg cells could out-compete polarized “proinflammatory” T-cell clones as well as naïve T cells. First, using the HY antigen-specific A1 system (H-2k), we asked whether nTreg cells from female A1 TCR transgenic mice [] would also behave competitively in aggregation around DCs. nTreg cells and naïve T cells were sorted from (A1.RAG−/− × CBA) F1 mice based on CD4 and CD25 expression. The T-cell populations were pretreated with either a green (PKH-67) or red (PKH-26) membrane dye prior to coincubation and incubated together at 1:1 ratio with bone marrow-derived DCs (BMDCs) and HYEk peptide (100 nM) for 48 h. IL-2, important for the function and maintenance of nTreg cells [], was also added to the cultures at 5 ng/mL. Naïve T cells were labeled with a green-membrane dye while nTreg cells were treated with the red dye (Fig. 1A top panel). After 24 h of coincubation, the cultures were then examined using confocal microscopy. Three random clusters were photographed in each culture condition, and a tally of the number of red and green cells in each cluster was made using ImageJ®.
Figure 1. nTreg cells out-compete naïve T cells in T cell-DC clusters. nTreg cells (CD4+CD25+) and naïve T cells (CD4+CD25−) (Tn) were sorted using Mo-Flo from (A1.RAG−/− × CBA) F1 mice on CD4 and CD25 expression. Cells were then labeled with membrane dyes (nTreg cells: red, Tn cells: green) and incubated together at 1:1 ratio with female BMDCs and HYEk peptide (100 nM) for 48 h after membrane dye treatment. IL-2 was also added to the cultures at 5 ng/mL. (A) Cells were transferred to glass-bottomed chamber slides and visualized using a Zeiss LSM 510 META laser scanning microscope. Three random clusters were photographed, and the stained cell populations in the clusters were quantified using ImageJ software®. Scale bars represent 20 μm. (B) Mean + SEM of n = 3 clusters is shown. p-value was <0.001 using unpaired Student t-test with two-tailed analysis. Results are representative of two independent experiments.
Download figure to PowerPoint
When the clusters were examined, nTreg cells showed a strong preferential aggregation around BMDCs compared to the naïve effector cells such that approximately 70% of cells in the clusters were nTreg cells (Fig. 1A bottom panel and B), thus confirming the data of Onishi et al. and Sarris et al. [, ].
Competitive aggregation of TGF-β-conditioned T cells and cTreg cells is not dependent on Foxp3 expression
Because we found that nTreg cells and TGF-β-conditioned T cells can out-compete naïve T cells around DCs, and activated T cells also display this ability, we next asked what role, if any, expression of Foxp3 had in endowing these cells with competitive clustering ability. To this end, we used retrovirally transduced conditional Treg (cTreg) cells (cFoxp3-transduced T cells treated with 4-hydroxytamoxifen in vitro). cTreg cells were derived by transducing CD4+ T cells isolated from CBA or Marilyn.RAG1−/−.Foxp3hCD2 (see Materials and methods). T cells transduced with cFoxp3 retrovirus expressed abundant GFP-tagged Foxp3 protein predominantly in the cytoplasm (Fig. 3A middle panel). Upon addition of 4-hydroxytamoxifen (4HT) the fluorescence translocated to the nucleus indicating the Foxp3 protein had entered the nucleus (Fig. 3A lower panel). Expression of Foxp3 increased following transduction reaching a peak after 64 h of culture (Fig. 3B). Transduced cells cultured in the presence of 4HT seemed to express approximately threefold more Foxp3 than those cultured without 4HT. (This may be a consequence of nuclear sequestering of Foxp3 reducing its exposure to cytoplasmic proteases.) We then tested the ability of 4HT to switch the cFoxp3-transduced cells to a regulatory phenotype. cFoxp3 transduced Marilyn.RAG−/− CD4+ T cells or empty vector control cells were mitomycin C treated to inhibit their proliferation prior to coincubation with untransduced Marilyn.RAG−/− CD4+ T cells and female mitomycin-treated C57BL/6 BMDCs with cognate peptide. cFoxp3-transduced T cells cultured with 4HT significantly inhibited the proliferation of naïve Marilyn.RAG−/− CD4+ T cells in cocultures (Fig. 3C). In the absence of either 4HT or translocated cFoxp3, effector cell proliferation was not significantly inhibited by these cells.
Figure 3. cFoxp3-transduced CD4+ T cells behave as Treg cells in the presence of 4-hydroxytamoxifen. (A) CD4+CD25− cells were purified from the spleen of a CBA mouse and transduced with cFoxp3. At 40 h GFP+ cells were sorted using flow cytometry and examined using confocal microscopy. The blue DRAQ5 dye stains the nuclei of these cells. In the absence of 4HT the DRAQ5 and GFP show separate localization as shown by the arrows. Upon addition of 4HT the Foxp3 has translocated to the nuclei causing colocalization of the DRAQ5 and GFP fluorescence highlighted by arrows (lower panel). Scale bars represent 20 μm. (B) The expression of Foxp3 was assessed by flow cytometry at 40, 64, and 88 h following the onset of transduction. Transduced cells were gated on live CD4+GFP+ cells. nTreg cells were gated on live CD4+ cells. Data shown are from one experiment representative of three independent experiments. (C) cFoxp3- or empty vector-transduced Marilyn.RAG−/− CD4+ cells (40 h transduction) were treated with mitomycin C and incubated with naïve Marilyn.RAG−/− CD4+ cells (effector cells) and DCs presenting their cognate peptide, with (black bars) or without (white bars) 4HT (50 nM). The proliferation of effector cells was assessed 72 h later and shown as mean + SEM of three independent samples, representative of three experiments. Each well of the 96-well plate contained 104 BMDCs, 2 × 104 suppressors and 2.5 × 104 effectors. Statistical analysis was performed using two-way ANOVA with Bonferroni posttest.
Download figure to PowerPoint
We examined the competitive clustering ability of these cTreg cells by coincubation with naïve CD4+ T cells together with B6 BMDCs and HYAb peptide. 4HT was also added in vitro to ensure functional “activation” of cFoxp3 (50 nM). As controls, empty vector-transduced CD4+ cells with 4HT, and also cFoxp3-transduced CD4+ cells without 4HT, were used. The various cell populations were incubated with naïve CD4+ cells at 2:1, 1:1, and 1:2 ratios (Fig. 4A). While cTreg cells could out-compete naïve CD4+ cells, both empty vector-transduced cells and cFoxp3-transduced CD4+ cells without 4HT treatment could also do the same. There was no evidence that the aggregation potency of cTreg cells was vastly superior to that of control-transduced cells at various cell ratios (Fig. 4A and B). Hence, functional Foxp3 may not be essential for the competitive aggregation of cTreg cells.
Figure 4. cTreg cells are not better at out-competing naïve T cells in aggregating around DCs than empty vector-transduced T cells or 4HT-nontreated cFoxp3-transduced T cells. (A) CD4+ cells from Marilyn.RAG1−/−.Foxp3hCD2 mice were transduced with cFoxp3 in the presence of 4HT (50 nM) for 40 h and then sorted by flow cytometry. After membrane dye treatment, CD4+GFP+ (cTreg) cells were then incubated with naïve sorted CD4+ (Tn) cells in the presence of B6 female BMDCs and 4HT (50 nM) at 2:1, 1:1, and 1:2 transduced cell:naïve CD4+ ratios. T cell-DC clusters were then examined with confocal microscopy. Empty vector-transduced T cells (treated with 4HT) and cFoxp3-transduced but 4HT-nontreated cells were also coincubated with naïve cells as controls. Scale bars represent 20 μM. (B) Four random clusters were quantified for each group at each cell ratio and shown as mean ± SEM. Data shown are representative of one of three separate experiments. Statistical analysis was performed using two-way ANOVA with Bonferroni posttest; see Supporting Information Fig. 1 for detailed statistical results.
Download figure to PowerPoint
To further investigate whether Foxp3 was important for competitive aggregation of iTreg cells, Foxp3+, and Foxp3- populations of TGF-β-conditioned T cells from Marilyn.RAG1−/−.Foxp3hCD2 mice [] were purified based on their expression of hCD2 and CD4 (Fig. 5A and B). hCD2+ (Foxp3+) and hCD2− (Foxp3−) cells were then coincubated with naïve T cells. At both 10:1 and 1:1 ratios, there was no statistically significant difference in the aggregation potency of hCD2+ and hCD2− cells compared to naïve T cells. This suggests that Foxp3+ TGF-β-conditioned T cells are no better at out-competing naïve T cells than Foxp3- TGF-β-conditioned T cells (Fig. 5C).
Figure 5. Foxp3+ TGFβ-conditioned T (iTreg) cells are no better at out-competing naïve T cells than Foxp3-TGF-β-conditioned T cells. (A) TGF-β-conditioned T cells from Marilyn.RAG1−/−. Foxp3hCD2 mice were sorted based on the expression of hCD2 and CD4. (B) The Foxp3 expression of CD4+hCD2− and CD4+hCD2+ cells are shown. (C) The hCD2− and hCD2+ subsets of TGF-β-conditioned cells were incubated with naïve T (Tn) cells in the presence of B6 female BMDCs for 24 h. Quantitation was then conducted on the T cell-DC clusters and shown as mean ± SEM of four random clusters, representative of one of three independent experiments. Statistical analysis was performed using two-way ANOVA with Bonferroni posttest; see Supporting Information Fig. 1 for detailed statistical results.
Download figure to PowerPoint
TGF-β-conditioned T cells and cTreg cells do not out-compete other activated T cells in aggregation around DCs
Having established that both regulatory cells and activated Foxp3− CD4+ T cells could out-compete naïve T cells, we hypothesized that previous antigen activation could be more important for the ability of T cells to competitively cluster around DCs. Onishi et al.  attributed the competitive aggregation of nTreg cells to their LFA-1 expression, as LFA-1-deficient nTreg cells fail to out-compete naïve T cells in aggregation. As LFA-1 is upregulated on T-cell activation [], this would be consistent with the observation that antigen-activated CD4+ T cells show superior competitive aggregation compared to naïve CD4+ T cells. Moreover, nTreg cells may also be more efficient than naïve CD4+ T cells, as they may have constant exposure to the repertoire of self-antigens [].
We tested whether there was a Treg cell-intrinsic superiority in competitive aggregation by comparing the competitive clustering ability of activated CD4+ T cells with CD4+ T cells transduced with cFoxp3 empty vector or cFoxp3 in the presence and absence of 4HT. Sorted cTreg cells (cFoxp3-transduced CD4+ T-cell, 4HT treated) were coincubated with untransduced but anti-CD3 activated CD4+ T cells for 24 h. Empty vector-transduced cells and non-4HT-treated cFoxp3-transduced cells were also coincubated with untransduced-activated cells for comparison. cTreg cells did not out-compete activated CD4+ T cells more so than control-transduced cells at the cell ratios that were examined — 2:1, 1:1, and 1:2 (Fig. 6).
Figure 6. cTreg cells do not out-compete-activated T cells in aggregation.CD4+ T cells from Marilyn.RAG1−/−.Foxp3hCD2 mice were transduced with cFoxp3 in the presence of 4HT (50 nM) for 40 h and then sorted by flow cytometry. CD4+GFP+ (cTreg) cells were then incubated with anti-CD3 activated but untransduced CD4+ (Ta) cells in the presence of B6 female BMDCs and 4HT (50 nM) at 2:1, 1:1, and 1:2 cell ratios. T cell-DC clusters were then examined with confocal microscopy. Empty vector-transduced T cells (treated with 4HT) and cFoxp3-transduced but 4HT-nontreated cells were also coincubated with untransduced cells as controls. Data are shown as mean + SEM of four aggregates and are representative of one of three independent experiments. Statistical analysis was performed using two-way ANOVA with Bonferroni posttest; see Supporting Information Fig. 1 for detailed statistical results.
Download figure to PowerPoint
Polarized T-cell clones also out-compete naïve T cells
While nTreg cells, iTreg cells, and cTreg cells all express Foxp3, they also share the common characteristic of being antigen experienced. nTreg cells are thought to constantly survey antigen in the periphery and cTreg cells and iTreg cells are induced during activation with cognate antigen. Prior antigen experience may explain the result that empty vector-transduced CD4+ T cells and Foxp3- TGF-β-conditioned cells can out-compete naïve T cells in aggregation equally well compared to cTreg cells and iTreg cells. To confirm if antigen experience is sufficient for competitive aggregation of CD4+ T cells, polarized cell clones (Th1, Th2, Tr1, Th9) were incubated with naïve CD4+ T cells (see Materials and methods on the generation of polarized cell clones). Both naïve CD4+ T cells and polarized cell clones were established from A1.RAG−/− mice. In contrast to naïve T cells, polarized T-cell clones had been activated for 14 days with the male spleen cells prior to the aggregation assay.
As seen in Fig. 7A, all polarized cell clones were found to out-compete naïve CD4+ T cells (Tn). This was statistically significant at all cell ratios for Th9 and Th2 cell clones, and at 2:1 and 1:1 cell ratios for Th1 and Tr1 cell clones. At the 1:1 ratio, Th9, Tr1, and Th1 appeared to be the most competitive at aggregating, with around 70% cells in the T cell‒DC clusters being red (polarized cell clone). Hence, antigen-experienced proinflammatory and regulatory cell types all appear to out-compete naïve antigen-inexperienced cells.
Figure 7. Polarized inflammatory cell clones out-compete naïve T cells in aggregating around DCs. (A) Polarized Th1, Th2, Tr1, and Th9 cell clones were labeled with the red membrane dye PKH-26 and naïve CD4+ T (Tn) cells isolated from A1.RAG−/− splenic cells were labeled with the green membrane dye PKH-67. Cells were incubated with female BMDCs and HYEk peptide (100 nM) for 24 h before being examined by confocal microscopy. Quantitation was performed by counting coloured aggregating cells in four random clusters. Data are representative of one of five independent experiments. Statistical analysis was performed using two-way ANOVA with Bonferroni posttest; see Supporting Information Fig. 1 for detailed statistical results. (B) Polarized Th1, Th2, Tr1, and Th9 cell clones and 7-day peptide-activated A1.RAG−/− CD4+ cells were labeled with the red membrane dye (PKH-26). TGF-β-conditioned T (TTGF-β-con) cells were labeled with the green membrane dye (PKH-67). The percentage of TGF-β-conditioned T cells expressing Foxp3 was 46%. Activated T cells were prepared in the same way as TGF-β-conditioned T cells, except TGF-β was not added to the culture. Data are shown as mean ± SEM of four aggregates and are representative of one of five independent experiments. Statistical analysis was performed using two-way ANOVA with Bonferroni posttest; see Supporting Information Fig. 1 for detailed statistical results. (C) TGF-β-conditioned T cells do not activate DCs to the same extent as proinflammatory polarized cell T clones. CBA female BMDCs were incubated with naïve T (Tn) cells, TGF-β-conditioned T (TTGF-β-con) cells and various polarized cell clones. DCs (CD11c+) were examined by flow cytometry at 24 and 48 h time points for CD86, CD40, and MHC class II expression. Data are shown as mean of three replicates and are representative of one of three independent experiments.
Download figure to PowerPoint
To examine the aggregation competitiveness of TGF-β-conditioned T cells in comparison with other antigen-experienced cell types, the former were coincubated with activated but non-polarized T cells and polarized cell clones at varying ratios. There was no statistically significant difference in the competitive aggregation of TGF-β-conditioned T cells with activated T cells compared with dye-control TGF-β-conditioned T-cell incubations (Fig. 7B). Furthermore, TGF-β-conditioned cells competed poorly in aggregation against the polarized cell clones Th1 and Tr1 (Fig. 7B). Hence, while cTreg cells and TGF-β-conditioned T cells compete favorably against naïve T cells, they do not appear to have a competitive advantage in relation to other antigen-experienced cell populations.
DCs are important for both the induction of primary immune responses and the promotion of immunological tolerance [[18, 19]]. Upon encounter with CD4+ T cells, DCs are activated via CD40-CD40L interaction, leading to the upregulation of CD80 and CD86 costimulatory molecules and release of cytokines. CD86 is one of the key molecules for the amplification of the T-cell response []. CD40 is also upregulated upon BMDC maturation and activation. To examine whether iTreg cells can modulate the expression of activation markers such as CD40 and CD86 on DCs, BMDCs were examined after 24 or 48 h culture with TCR-transgenic TGF-β-conditioned T cells in the presence of cognate peptide (100 nM). For comparison, naïve T cells and polarized T-cell lines were also coincubated with BMDCs (Fig. 7C).
BMDCs coincubated with Th1 and Th9 polarized CD4+ cells for 24 h were found to express high levels of CD86 (MFI: 584 and 326, respectively), with much lower levels found on those coincubated with Th2, Tr1, naive, and TGF-β-conditioned T cells (MFI: 71, 63, 59, and 87) at 24-h postincubation (Fig. 7C). The same is also true at 48 h. Similarly, at 24 h, CD40 was significantly upregulated by BMDCs coincubated with Th1 and Th9 polarized cell clones (MFI: 609 and 561), but lower levels were found on those coincubated with TGF-β-conditioned cells, Th2, Tr1, and naive cells (MFI: 15, 188, 268, 20). A similar picture also emerged for the expression of class II MHC on BMDCs, with Th1 and Th9 polarized cells producing the highest levels of expression and lower levels produced by TGF-β-conditioned cells, Tr1 cells, and naïve T cells.
Hence, antigen-experienced cells such as TGF-β-conditioned T cells and polarized CD4+ clones, while effective in competitive aggregation compared with naïve T cells, are not equal in their ability to induce upregulation of activation markers CD86, CD40, and class II MHC on BMDCs. TGF-β-conditioned T cells appear less effective at activating BMDCs than polarized inflammatory cell types such as Th1 and Th9 cells. Taken together, TGF-β-conditioned T cells can not only physically displace naïve T cells from contact with DCs but also only modestly activate the DCs themselves. Like “civil servants,” these physically competitive but functionally inert cells may contribute to the dampening of the immune response in an inflammatory context by limiting the capacity of naïve CD4+ T cells to bind to DCs [].