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DOI: 10.1002/eji.201343463
Eur. J. Immunol. 2014. 44: 460–468
Induced and thymus-derived Foxp3+ regulatory T cells share a common niche Yi-Ju Huang1 , Verena Haist2 , Wolfgang Baumg¨ artner2 , Lisa F¨ ohse3 , Immo Prinz3 , Sebastian Suerbaum4 , Stefan Floess∗1 and Jochen Huehn∗1 1
Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Department of Pathology, University of Veterinary Medicine, Hannover, Germany 3 Institute of Immunology, Hannover Medical School, Hannover, Germany 4 Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Foxp3+ regulatory T (Treg) cells, which play a central role for the maintenance of immune homeostasis and self-tolerance, are known to be both generated in the thymus (thymusderived, tTreg cells) and in the periphery, where they are converted from conventional CD4+ T cells (induced Treg (iTreg) cells). Recent data suggest a division of labor between these two Treg-cell subsets since their combined action was shown to be essential for protection in inflammatory disease models. Here, using the transfer colitis model, we examined whether tTreg cells and iTreg cells fill different niches within the CD4+ T-cell compartment. When naive T cells were co-transferred with either pure tTreg cells or with a mixture of tTreg cells and iTreg cells, induction of Foxp3+ Treg cells from naive T cells was not hampered by preoccupation of the Treg-cell niche. Using neuropilin-1 (Nrp1) as a surface marker to separate tTreg cells and iTreg cells, we demonstrate that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and display comparable TCR repertoires. However, when transferred together Nrp1+ tTreg cells outcompeted Nrp1− iTreg cells and dominated the Treg-cell compartment. Taken together, our data suggest that tTreg cells and iTreg cells share a common peripheral niche.
Keywords: Colitis r iTreg cells
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Neuropilin-1 r Treg cells
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tTreg cells
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction Regulatory T (Treg) cells, expressing the lineage specification factor Foxp3, play a central role in immune tolerance to self and innocuous foreign antigens [1]. The majority of Foxp3+ Treg cells is generated during thymic development (thymus-derived, tTreg cells) and is believed to be selected for recognition of self-antigens.
Correspondence: Prof. Jochen Huehn e-mail: [email protected] C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In the periphery, the Treg-cell repertoire needs to be complemented by specificities directed against nonpathogenic foreign antigens, including commensal microbiota and food antigens, and this expansion of the Foxp3+ Treg-cell pool is achieved by peripheral conversion of conventional Foxp3− CD4+ T cells into Foxp3+ Treg cells (induced Treg (iTreg) cells) [2–5]. Recently, Helios, a member of the Ikaros transcription factor family, was
∗
These authors share senior co-authorship.
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suggested as a marker to distinguish between tTreg cells and iTreg cells [6, 7], however Helios has also been associated with T-cell activation and differentiation, and under certain conditions can be upregulated in iTreg cells [3, 8, 9]. More recently, the cell surface receptor neuropilin-1 (Nrp1), which was initially identified in a microarray-based approach as a Treg-cell-specific molecule [10], was reported to discriminate between Nrp1+ tTreg cells and Nrp1− iTreg cells particularly under steady-state conditions [11, 12]. Several studies have demonstrated that iTreg cells are capable of suppressing autoimmune responses to a similar degree as tTreg cells [2, 3, 13] and play a central role for the acquisition of mucosal as well as fetomaternal tolerance [14, 15]. Interestingly, full suppression in these autoimmune disease models was only achieved in the combined presence of both tTreg cells and iTreg cells [3, 13], and this complementary action was explained by the unique TCR repertoire of iTreg cells, which expanded the TCR diversity of the total Treg-cell pool [3, 4]. In the present study, we asked whether tTreg cells and iTreg cells occupy separate niches or whether they can supplement each other and fill a common Treg-cell niche. By performing adoptive transfer studies into lymphopenic recipient mice, we demonstrate that de novo induction of Foxp3+ Treg cells is not prevented by the presence of iTreg cells, that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and that tTreg cells dominate over iTreg cells when transferred under competitive conditions.
Results Preoccupation of the Treg-cell niche does not influence de novo generation of Foxp3+ Treg cells It is widely accepted that a division of labor exists between tTreg cells and iTreg cells and that both subsets are essential for the suppression of inflammatory immune responses in various tissues [2, 13, 15–17]. Nevertheless, it is only incompletely understood if de novo generation of iTreg cells is influenced by a preoccupation of the peripheral Treg niche. To address this question, we used the T-cell transfer colitis model, in which adoptive transfer of naive Foxp3− CD4+ T cells into immunodeficient recipient mice results in severe intestinal inflammation a few weeks after transfer, which can be prevented by co-transfer of Foxp3+ Treg cells [18]. Importantly, under these lymphopenic conditions, a significant fraction of transferred naive T cells converts into Foxp3+ iTreg cells in situ [19], and these iTreg cells were shown to play an important role for the full prevention of colitis [13]. Here, congenically marked naive Thy1.1+ T cells were adoptively transferred into SCID recipient mice either alone or co-transferred with a low (ratio 8:1) or a high (ratio 1:1) number of Thy1.1− Foxp3+ Treg cells. Five weeks after transfer, de novo generated iTreg cells could be detected among Thy1.1+ CD3+ CD4+ cells isolated from colonic lamina propria (cLP), mesenteric lymph node (mLN) and skin-draining, peripheral lymph node (pLN) (Fig. 1A and B), without observing a preferential iTreg-cell generation at mucosal sites as had been reported before [5, 19]. De novo generation of iTreg C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. De novo induction of Treg cells is independent of the presence of co-transferred Treg cells. Naive T cells (Thy1.1+ ) were adoptively transferred alone (1:0 ratio) or together with CD4+ Foxp3GFP+ Treg cells (Thy1.1− ) isolated from spleens and LN of DEREG mice into SCID recipients at indicated ratios. (A) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ -gated cLP lymphocytes isolated from recipient mice 5 weeks after transfer. Numbers indicate frequency of cells within quadrants. (B) The frequency of de novo induced Foxp3+ cells among total Thy1.1+ CD3+ CD4+ cells and (C) the frequency of total Foxp3+ Treg cells among CD3+ CD4+ cells in pLN, mLN, and cLP of indicated groups are shown as mean + SD of n = 5–6 mice from one experiment representative of two independent experiments. n.s.: not significant, *p < 0.05, **p < 0.01, Kruskal–Wallis and Mann–Whitney test.
cells not only occurred in SCID mice receiving naive T cells alone but also in mice that received naive T cells together with Treg cells (Fig. 1A). Strikingly, the frequency of de novo generated iTreg cells (Thy1.1+ Foxp3+ ) was largely independent of the presence of co-transferred Thy1.1− Treg cells and the ratio between naive T cells and Thy1.1− Treg cells (Fig. 1B). Comparable results were achieved when a higher cell number was used for adoptive transfer of naive T cells and Treg cells at a ratio of 8:1 (Supporting Information Fig. 1). The absolute number of de novo generated iTreg cells (Supporting Information Fig. 2A) and the frequency of total Foxp3+ Treg cells (Fig. 1C) was influenced by the number of initially co-transferred Treg cells, but no significant differences between total Treg-cell numbers between the different groups were observed (Supporting Information Fig. 2B). These findings can be best explained by the fact that co-transferred Treg cells not only affected total lymphocyte numbers (Supporting Information Fig. 2C), but also the strength of the inflammatory response within the colon (Supporting Information Fig. 3). Together, our results www.eji-journal.eu
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Figure 2. De novo induction of Foxp3+ Treg cells is not impaired in the presence of iTreg cells. Naive T cells (Thy1.1+ ) were adoptively transferred together with CD4+ Foxp3GFP+ Treg cells (Thy1.1− ) isolated from thymus (thymic-Treg), mLN (mLN-Treg) or pLN (pLN-Treg) of DEREG mice into SCID recipients at a ratio of 8:1. (A) Representative flow cytometry data showing Helios expression among CD4+ Foxp3GFP+ Treg cells isolated from thymus, mLN or pLN of DEREG mice. Numbers indicate frequency of Helios+ cells and shaded histograms represent isotype controls. (B) The frequency of Helios+ (black bar) and Helios− (white bar) cells among CD4+ Foxp3GFP+ cells from the indicated organs are shown as mean + SD of n = 3 from one experiment representative of three independent experiments performed. (C) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ cLP lymphocyte isolated from recipient mice 10 weeks after transfer. Numbers indicate frequency of cells within quadrants. (D) The frequency of de novo induced Foxp3+ cells among total Thy1.1+ CD3+ CD4+ cells and (E) the proportion of Thy1.1+ (dashed bars) and Thy1.1− (filled bars) Foxp3+ Treg cells among CD3+ CD4+ cells isolated from pLN, mLN, and cLP of indicated groups are shown as mean + SD of n = 6 from one experiment representative of two independent experiments performed. n.s., not significant, Kruskal–Wallis test.
show that de novo induction of Treg cells is not prevented but modulated by the preexistence of Foxp3+ Treg cells.
De novo induction of Foxp3+ Treg cells is not influenced by presence of iTreg cells Our finding that co-transfer of total Treg cells does not prevent de novo generation of iTreg cells did not answer the question if iTreg cells do occupy a unique niche that is different from the niche for tTreg cells and if the preexistence of iTreg cells influences de novo generation of Treg cells from naive T cells. To address this issue, Foxp3+ Treg cells were isolated from thymi, mLN, or pLN of Thy1.1− donor mice and co-transferred together with Thy1.1+ naive T cells into SCID recipient mice. Treg cells isolated from the thymus can be regarded as a largely pure tTreg-cell population, showing the highest frequency of Helios+ cells (Fig. 2A and B) and containing only scarce recirculating peripheral Treg cells [20]. In contrast, mLN- and pLN-derived Treg cells are known to contain a substantial fraction of iTreg cells, which are believed to reside mainly within the Helios− fraction (Fig. 2A and B). Ten weeks after transfer, histopathological analyses of colonic tissues revealed no significant differences in the strength of C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the inflammatory response between groups of mice receiving thymus-, mLN- or pLN-derived Treg cells (Supporting Information Fig. 4). Interestingly, frequencies and absolute numbers of de novo generated iTreg cells (Thy1.1+ Foxp3+ ) within pLN, mLN, and cLP were comparable between the different groups (Fig. 2C and D and Supporting Information Fig. 5A), suggesting that the preexistence of iTreg cells does not significantly hamper de novo generation of iTreg cells. Moreover, 10 weeks after transfer frequencies of total Treg cells as well as absolute numbers of total Treg cells and total lymphocytes were comparable within all organs analyzed between groups of mice receiving thymus-, mLN- or pLN-derived Treg cells (Fig. 2E and Supporting Information Fig. 5B and C), suggesting that the size of the total Treg-cell compartment in lymphopenic recipient mice is independent of the origin of transferred Treg cells. The results obtained so far suggested that iTreg cells do not occupy a unique niche, but rather share a common niche with tTreg cells. To exclude that the fraction of iTreg cells within mLN-Treg cells or pLN-Treg cells was too small to significantly influence de novo generation of iTreg cells from naive T cells, we next used the recently described cell surface marker Nrp1 [11, 12] to isolate iTreg cells (Nrp1− ) and tTreg cells (Nrp1+ ) from spleens of healthy Thy1.1− mice for co-transfer experiments.
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Figure 3. Co-transfer of Nrp1− iTreg cells does not affect de novo generation of iTreg cells and results in a completely filled Treg-cell niche. Naive T cells (Thy1.1+ ) were adoptively transferred together with Nrp1+ or Nrp1− CD4+ Foxp3GFP+ Treg-cell subsets (Thy1.1− ) isolated from spleens of DEREG mice into SCID recipients at a ratio of 8:1. (A) Representative flow cytometry data show Nrp1 versus Foxp3GFP expression on gated CD4+ splenocytes before sort (left) as well as sort purity and Helios expression within sorted Treg-cell subsets (right). Numbers indicate frequency of gated cells and shaded histograms represent isotype controls. (B) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ gated cLP lymphocyte isolated from recipient mice 10 weeks after transfer. Numbers indicate frequency of cells within quadrants (C) The stability of Foxp3 expression in transferred Thy1.1− Foxp3+ Treg cells (gated on total Thy1.1− CD3+ CD4+ cells), (D) the frequency of de novo induced Foxp3+ cells among total Thy1.1+ CD3+ CD4+ cells and (E) the proportion of Thy1.1+ (dashed bars) and Thy1.1− (filled bars) Foxp3+ Treg cells among CD3+ CD4+ cells in pLN, mLN, and cLP of indicated groups are shown as mean + SD of n = 11 pooled from two independent experiments. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Mann–Whitney test.
Sorted Foxp3+ Nrp1+ tTreg cells homogeneously expressed Helios and Foxp3+ Nrp1− iTreg cells were largely Helios− (Fig. 3A). Ten weeks after transfer, histopathological analyses of colonic tissues revealed no significant differences in the strength of the inflammatory response between groups of mice receiving Nrp1+ or Nrp1− Treg cells (Supporting Information Fig. 6). As expected from the results of a recent study introducing Nrp1 as a marker to discriminate between tTreg cells and iTreg cells [11, 12], Nrp1+ tTreg cells showed a rather stable Foxp3 expression, whereas Nrp1− iTreg cells had substantially lost Foxp3 expression in pLN, mLN, and cLP 10 weeks after transfer (Fig. 3B and C). We next determined the frequency of de novo generated iTreg cells (Thy1.1+ Foxp3+ ). Surprisingly, we observed a significantly higher iTreg generation in pLN, mLN, and cLP of mice being cotransferred with Nrp1− iTreg cells compared to mice receiving Nrp1+ tTreg cells (Fig. 3B and D), and a significantly higher absolute number of iTreg cells was found in the cLP of mice that had received Nrp1− iTreg cells when compared to mice that had received Nrp1+ tTreg cells (Supporting Information Fig. 7A). Importantly, frequencies of total Treg cells (Fig. 3E) as well as C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
absolute numbers of total Treg cells and total lymphocytes were not different between both groups of mice within all organs analyzed (Supporting Information Fig. 7 B and C), showing that both Nrp1− iTreg cells and Nrp1+ tTreg cells could uniformly fill the total Treg niche. Viewed as a whole, our data strongly argue against the existence of a unique iTreg-cell niche, but support the notion that iTreg cells rather share a common niche with tTreg cells in the periphery.
Nrp1+ tTreg cells outcompete Nrp1− iTreg cells to dominate the Treg-cell compartment High diversity of the TCR repertoire is of utmost importance for the homeostasis and function of Treg cells [21]. Our finding that Nrp1+ tTreg cells and Nrp1− iTreg cells are equally potent to uniformly fill the entire Treg-cell niche in lymphopenic mice suggested that both Treg-cell subsets displayed a comparable TCR repertoire. Thus, we analyzed the TCR repertoire in Nrp1+ and Nrp1− Treg cells isolated from spleens of Foxp3 reporter mice
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Figure 4. TCR repertoire of Nrp1+ tTreg and Nrp1− iTreg subsets. CD4+ T cells from spleens of Thy1.1+ Foxp3hCD2 mice were sorted into Nrp1+ Treg cells (black), Nrp1− Treg cells (white), and Foxp3− cells (gray) and their Tcra repertoire was analyzed by next-generation sequencing. The Jα usage of functionally rearranged TRAV12 (Vα8) family sequences for each population is shown as mean + SD of four individual samples from one experiment.
(Fig. 4). Analysis of the TRAV12 (Vα8) family, which constitutes approximately 10% of all rearranged TCR-α chains in murine CD4+ T cells, revealed an overall similar J usage pattern between both Treg-cell subsets and even Foxp3− CD4+ T cells (Fig. 4). A comparable degree of sequence overlap among all three populations was also evident when comparing similarities by Morisita–Horn indices of the respective CDR3 regions (Supporting Information Fig. 8A and B). This indicates that Nrp1+ and Nrp1− Treg cells used in the former experiment (Fig. 3) were equipped with similar TCR repertoires. Having shown that Nrp1+ tTreg cells and Nrp1− iTreg cells can both uniformly fill the entire Treg-cell niche and display similar TCR repertoires, we next asked how these two Tregcell subsets behave under competitive conditions. To this end, we adoptively transferred Thy1.1+ naive T cells together with 4:1 or 1:4 mixtures of Nrp1+ tTreg cells and Nrp1− iTreg cells into Rag2-deficient mice (Fig. 5A). The Treg-cell subsets were Thy1.1− and either CD45.1+ or CD45.2+ to allow definite identification. A swap of donor mice was included in the experimental setup to avoid a strain-driven bias. Eight weeks after adoptive transfer, histopathological analyses of colonic tissues revealed no significant differences in the strength of the inflammatory response between groups of mice receiving 4:1 or 1:4 mixtures of Nrp1+ tTreg cells and Nrp1− iTreg cells (Supporting Information Fig. 9A). Furthermore, frequencies and absolute numbers of de novo generated iTreg cells (Thy1.1+ Foxp3+ ) within pLN, mLN, and cLP were comparable between the two groups (Supporting Information Fig. 9B and C). Strikingly, when we analyzed the composition of the Thy1.1− Foxp3+ Tregcell compartment, we observed that the tTreg-cell progeny dominated the Treg-cell compartment in both groups receiving a mixture of Treg cells (Fig. 5A and B). Absolute numbers of Thy1.1− Foxp3+ cells and total lymphocytes did not show any significant differences between the two groups at different locations (Supporting Information Fig. 9D and E). In summary, these results demonstrate that tTreg cells can outcompete iTreg cells if transferred together and dominate the total Treg-cell niche at the endpoint of the experiment.
Discussion The peripheral pool of Foxp3+ Treg cells is a heterogeneous population of tTreg cells and iTreg cells. In the present study, we asked whether tTreg cells and iTreg cells occupy separate niches C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. Nrp1+ tTreg cells outcompete Nrp1− iTreg cells and dominate the Treg-cell compartment. Naive T cells (Thy1.1+ ) were adoptively transferred together with a mixture of Nrp1+ and Nrp1− CD4+ Foxp3+ Treg-cell subsets (Thy1.1− ) into RAG2−/− recipients at a ratio of 8:1. The Treg-cell mixture consisted of Nrp1+ and Nrp1− Treg cells isolated from spleens of FIR (CD45.2+ ) or Foxp3hCD2 Rag1GFP (CD45.2− ) mice at a ratio of 4:1 or 1:4. (A) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ gated cells (left) and CD45.2 expression in CD3+ CD4+ Thy1.1− Foxp3+ gated cells (right) isolated from mLN of recipient mice 8 weeks after transfer. Numbers indicate frequency of cells within quadrants or gated cells. (B) The frequency of Nrp1+ progeny among CD3+ CD4+ Thy1.1− Foxp3+ cells in pLN, mLN, and cLP of indicated groups is shown as mean ± SD of n = 7–8 pooled from two independent experiments.
or whether they can supplement each other and fill a common Treg-cell niche. By performing adoptive transfer studies into lymphopenic recipient mice, we could demonstrate that de novo induction of Foxp3+ Treg cells is not prevented by the presence of iTreg cells, that tTreg cells and iTreg cells share a common Tregcell niche in the periphery and that tTreg cells dominate over iTreg cells if transferred under competitive conditions. Adoptive transfer of naive Foxp3− CD4+ T cells into lymphopenic recipient mice is known to result in severe intestinal inflammation a few weeks after transfer [18]. Although previous in vitro studies have reported that de novo induction of Foxp3+ Treg cells is negatively influenced by inflammatory cytokines [22–24], in the present study, we observed efficient iTreg-cell generation even in mice that did not receive any co-transferred Treg cells and www.eji-journal.eu
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that developed severe colitis 5 weeks after T-cell transfer. A scenario in which de novo induction of Foxp3+ Treg cells precedes the development of colitis in the recipient mice can best explain this discrepancy. Indeed, an impaired de novo induction of Foxp3+ Treg cells had been reported when sublethally irradiated mice, which immediately suffer from irradiation-induced inflammation, were used as recipients [25]. The functional properties of iTreg cells in the transfer colitis model are currently controversially discussed. Although a recently published paper showed that early development of iTreg cells could alleviate intestinal inflammation [26], in the present study, we observed no correlation of the frequency of iTreg cells with the colitis score. In line with our data, Izcue et al. reported a similar degree of colitis regardless whether the lymphopenic recipient mice did receive Foxp3-deficient or -sufficient naive T cells [27]. Despite these discrepancies, all studies agree on the fact that the local inflammatory milieu and the severity of the disease critically influence the functional properties of iTreg cells. Previous studies have reported a high degree of similarity between iTreg cells and tTreg cells as they show both similar gene expression profiles [3, 14] as well as comparable epigenetic signatures [28]. However, differences in the TCR repertoire seem to be dependent on the location of the Treg cells, especially the TCR repertoire of colonic Treg cells might be microbiota driven and therefore different from others [4]. In the present study, Nrp1+ tTreg cells and Nrp1− iTreg cells used for adoptive transfer experiments were isolated from the spleen, a place that is known to contain Treg cells with a less diverse TCR repertoire [29]. Accordingly, we did observe comparable TCR repertoires in Nrp1+ tTreg cells and Nrp1− iTreg cells, but presumably those TCR repertoires develop differently after transfer into lymphopenic hosts as it was recently published [3]. Nrp1 has been recently reported to discriminate between tTreg cells and iTreg cells, particularly under steady-state conditions [11, 12]. Although Nrp1 expression was shown to be influenced by TGF-β and inflammatory environments [3,11], this cell surface marker correlates to a high degree with the intracellular tTregcell marker Helios when healthy SPF-housed mice were taken as donors. After transfer of Nrp1+ tTreg cells or Nrp1− iTreg cells together with naive T cells into lymphopenic recipients, we found that both Treg-cell subsets could uniformly fill the entire Treg-cell niche. This is particularly worth mentioning in case of adoptive transfer of Nrp1− iTreg cells together with naive T cells since under these conditions tTreg cells are completely lacking in the recipient mice. Importantly, we here observed an even higher iTreg generation compared to mice receiving Nrp1+ tTreg cells, but this somewhat unexpected observation can be best explained by differences in the stability of Foxp3 expression between Nrp1− iTreg cells and Nrp1+ tTreg cells after transfer into immunodeficient mice. Indeed, Nrp1+ tTreg cells were recently reported to display a more complete demethylation of the foxp3 locus compared to Nrp1− iTreg cells [11], a phenotype that is known to correlate with stability of Foxp3 expression [30]. These data strongly argue against the existence of unique subniches for iTreg cells and tTreg C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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cells and is somewhat contrary to recently published data analyzing a mouse mutant that lacks the conserved noncoding sequence 1 (CNS1) of the foxp3 locus, a TGF-β − responsive element that has been suggested to play an important role for the generation of iTreg cells [14]. Co-transfer of Foxp3+ Treg cells from CNS1deficient mice together with naive T cells into lymphopenic recipient mice resulted in a higher de novo generation of iTreg cells when compared with that after co-transfers using Foxp3+ Treg cells from WT mice [14]. However, CNS1−/− mice still contained Nrp1− Foxp3+ Treg cells [11], suggesting that a significant fraction of iTreg cells can be generated independently of TGF-β. It thus still remains to be clarified whether TGF-β-dependent and -independent iTreg cells occupy unique subniches within the total Treg-cell compartment. The ability of tTreg cells and iTreg cells to uniformly fill the entire Treg niche raised the question about the relationship between these subsets. In view of the fact that there is a high degree of similarity in the gene expression profiles [3, 14], epigenetic signatures [28] and TCR repertoires (present study), it was somewhat unexpected to see a clear dominance of tTreg cells above iTreg cells in competitive co-transfer experiments, even when the majority of the transferred cells originate from Nrp1− iTreg cells. This finding can be explained by a different migratory behavior of Nrp1+ tTreg cells and Nrp1− iTreg cells, as recently reported in a tumor model using mice that lack Nrp1 selectively on CD4+ T cells [31]. Alternatively, ligation of Nrp1 on the surface of tTreg cells by its ligand semaphorin-4a can modulate Akt-mTor signaling, which finally promotes Treg-cell stability by enhancing quiescence, increasing expression of survival factors, slightly increasing proliferation, and concomitantly repressing terminal differentiation [32]. Together, these changes can explain our observation that Nrp1+ tTreg cells outcompete Nrp1− iTreg cells and dominate the entire Treg-cell niche when transferred together. In conclusion, the findings of the present study suggest that peripheral de novo induction of Foxp3+ Treg cells from naive T cells in lymphopenic models is not hampered by the preexistence of iTreg cells. An empty Treg-cell niche can be entirely filled by either tTreg cells or iTreg cells, providing strong arguments for a common Treg-cell niche in the periphery.
Materials and methods Mice DEREG mice [33], congenic BALB/c × Thy1.1+ mice, congenic BALB/c × Thy1.1+ Foxp3hCD2 mice ([34], kindly provided by Shohei Hori, Yokohama, Japan), Rag2-deficient C57BL/6 mice (C57BL/6 background), Foxp3IRES-RFP mice ([25], C57BL/6 background, kindly provided by Richard Flavell, New Haven, CT, USA), CD45.1+ Foxp3hCD2 Rag1GFP mice ([20], C57BL/6 background, kindly provided by Shohei Hori, Yokohama, Japan) and congenic www.eji-journal.eu
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C57BL/6J Thy1.1+ mice were bred under specific pathogen-free conditions at the animal facility of the Helmholtz Centre for Infection Research (Braunschweig, Germany). SCID mice and Thy1.2+ BALB/c mice were purchased from Charles River Germany. Animal care and all procedures were performed in accordance with institutional, state, and federal guidelines.
Nrp1− iTreg cells (tTreg:iTreg ratio 4:1) isolated from spleens of Foxp3IRES-RFP or CD45.1+ Foxp3hCD2 Rag1GFP . In one experiment, 9 × 105 naive T cells were adoptively transferred together with 1.125 × 105 CD4+ Foxp3hCD2+ Treg cells into SCID recipient mice. Body weight of recipients was monitored regularly, and mice were sacrificed when losing 20% of highest body weight.
Antibodies, staining, and flow cytometry
Purification of lamina propria lymphocytes
The following antibodies were purchased from eBioscience: antiCD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), antiCD25 (PC61.5), anti-CD11c (N418), anti-CD90.1 (HIS51), antiCD90.2 (53–2.1), anti-CD45R (RA3–6B2), anti-CD103 (2E7), anti-GITR (DTA-1), anti-Foxp3 (FJK-16s). Anti-hCD2 (RPA-2.10), anti-CD45.2 (104), anti-CD45RB (C363.16A), and anti-Helios (22F6) were purchased from Biolegend and biotinylated antiNrp1 (polyclonal) was purchased from R&D. Cells were stained with LIVE/DEAD Fixable Dead Cell stains (Invitrogen), followed by fluorochrome-conjugated surface marker staining. Cells were fixed, permeabilized, and stained intracellularly for Helios and Foxp3 using the Foxp3 staining set (eBioscience). Samples were acquired with an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Lymphocytes from large intestines of recipient mice were prepared as previously described [7] with minor modifications. Briefly, tissues were digested in Liberase Blendzyme (0.1 U/mL, Roche) and DNase I (0.1 mg/mL, Sigma) containing RPMI 1640 (Gibco) buffer for 45 min at 37◦ C followed by a 40%:70% Percoll gradient (Easycoll, Biochrom AG). Lamina propria lymphocytes were obtained from the interface of the gradient and resuspended in PBS buffer for further staining.
Cell sorting and adoptive transfer Naive CD4+ T cells were isolated from spleen and LN singlecell suspensions of 6–8 week old Thy1.1 mice by first enriching CD4+ cells using anti-CD4 microbeads and the autoMACS magnetic separation system (Miltenyi Biotec). Enriched CD4+ cells were stained with anti-CD4, anti-CD45RB, anti-GITR antibodies and the exclusion marker antibodies anti-CD8, antiCD11c, anti-B220, anti-CD25, and anti-CD103, and sorted into naive CD4+ CD45RBhigh GITRlow T cells, negative for the exclusion markers, by fluorescence-activated cell sorting (FACS) (FACSAria, BD Biosciences). For the isolation of organ-specific CD4+ Foxp3GFP+ Treg cells, CD4+ cells were first enriched from pLN, mLN, and thymic single-cell suspensions of 6–8 week old DEREG mice by anti-CD4 microbeads (pLN and mLN) or CD8Dynabeads (Invitrogen) to deplete CD8+ thymocytes. Enriched CD4+ T cells were stained with anti-CD4 and sorted into CD4+ Foxp3GFP+ Treg cells by FACS. For the isolation of Nrp1+ and Nrp1− CD4+ Foxp3+ Treg subsets, CD4-enriched splenocytes from DEREG, Foxp3IRES-RFP or CD45.1+ Foxp3hCD2 Rag1GFP mice were stained with anti-CD4, anti-Nrp1 and anti-hCD2, and sorted by FACS. Purity of sorted cells generally was ∼ 99%. 1.8 × 105 naive T cells were adoptively transferred by i.p. injection either alone (1:0 ratio) or together with 1.8 × 105 (1:1 ratio) or 2.25 × 104 (8:1 ratio) sorted CD4+ Foxp3GFP+ Treg cells derived from DEREG mice or a congenically marked Treg-cell mixture into SCID or Rag2−/− recipient mice. The Treg mixture consisted of either 4.5 × 103 Nrp1+ tTreg cells plus 1.8 × 104 Nrp1− iTreg cells (tTreg:iTreg ratio 1:4) or 1.8 × 104 Nrp1+ tTreg cells plus 4.5 × 103 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
TCR amplification and high-throughput sequencing Total RNA was isolated using the RNeasy Kit (Qiagen) from 70 000 FACS-sorted CD4+ Foxp3+ Nrp1+ , CD4+ Foxp3+ Nrp1− , or CD4+ Foxp3− cells, from four individually analyzed donors. cDNA templates were synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche) according to manufacturer’s recommendation. Amplicon libraries of rearranged TRAV12 (Vα8 family) CDR3 regions for the Genome Sequencer FLX System (454 sequencing, Roche) were generated as described before [21]. Amplicons were purified by agarose gel electrophoresis and QIAquick Gel Extraction Kit (Qiagen), and quantified by QuantiTTM dsDNA HS Assay Kit (Invitrogen). Sequencing reactions were performed by ultra-deep 454 pyrosequencing on the Genome Sequencer FLX system (Roche Applied Sciences) according to manufacturer’s recommendation. Productive rearrangements, J usage, and CDR3α regions were defined by comparing nucleotide sequences to the reference sequences from IMGTs, the international ImMunoGeneTics information systems (http://www.imgt.org; IMGT/HighV-QUEST) [35, 36].
Histology Two pieces from the colon of each recipient mouse were dissected, fixed in formalin, and embedded in paraffin. Tissue slices were stained with hematoxylin and eosin. Blinded sections of colon pieces were examined and classified into a five-level score [37].
Statistics Values generated by adoptive transfer experiments were analyzed by GraphPad Prism (GraphPad Software) with mean ± SD and calculated by Mann–Whitney test or Kruskal–Wallis test. P www.eji-journal.eu
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values < 0.05 were considered as statistically significant; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. For the analysis of the TCR repertoire, the similarity between two sequenced TCR repertoires was statistically measured by the Morisita–Horn index [38]. This index ranges between 0 (completely dissimilar) and 1 (identical) and is comparatively resistant to sample size. To compare all samples the cut off was set to 6700 productive sequences per sample.
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T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression. PLoS One 2011. 6: e20731. 10 Bruder, D., Probst-Kepper, M., Westendorf, A. M., Geffers, R., Beissert, S., Loser, K., von Boehmer, H. et al., Neuropilin-1: a surface marker of regulatory T cells. Eur. J. Immunol. 2004. 34: 623–630. 11 Weiss, J. M., Bilate, A. M., Gobert, M., Ding, Y., Curotto de Lafaille, M. A., Parkhust, C. N., Xiong, H. et al., Neuropilin-1 is expressed on thymusderived natural regulatory T cells, but not mucosa-generated induced Foxp3+ Treg cells. J. Exp. Med. 2012. 209: 1723–1742. 12 Yadav, M., Louvet, C., Davini, D., Gardner, J. M., Martinez-Llordella, M., Bailey-Bucktrout, S., Anthony, B. A. et al., Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J. Exp. Med. 2012. 209: 1713–1722. 13 Haribhai, D., Lin, W., Edwards, B., Ziegelbauer, J., Salzman, N. H., Carl-
Acknowledgements: We thank Lothar Gr¨ obe, Maria Ebel, Beate Pietzsch, and Sabrina Woltemate for excellent technical assistance. We thank Shohei Hori (Yokohama, Japan) and Richard Flavell (New Haven, CN) for providing Foxp3 reporter mice. This work was supported by a grant from the German Research Foundation (SFB621), the Hannover Biomedical Research School (HBRS) and the Center for Infection Biology (ZIB).
son, M. R., Li, S. H. et al., A central role for induced regulatory T cells in tolerance induction in experimental colitis. J. Immunol. 2009. 182: 3461– 3468. 14 Josefowicz, S. Z., Niec, R. E., Kim, H. Y., Treuting, P., Chinen, T., Zheng, Y., Umetsu, D. T. et al., Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 2012. 482: 395–399. 15 Samstein, R. M., Josefowicz, S. Z., Arvey, A., Treuting, P. M. and Rudensky, A. Y., Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell 2012. 150: 29–38.
Conflict of interest: The authors declare no financial or commercial conflict of interest.
16 Tannock, G. W., What immunologists should know about bacterial communities of the human bowel. Semin. Immunol. 2007. 19: 94–105. 17 Amsen, D., Spilianakis, C. G. and Flavell, R. A., How are T(H)1 and T(H)2 effector cells made? Curr. Opin. Immunol. 2009. 21: 153–160.
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Full correspondence: Prof. Jochen Huehn, Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany Fax: +49-531-6181-3399 e-mail: [email protected] Received: 19/2/2013 Revised: 2/9/2013 Accepted: 1/10/2013 Accepted article online: 8/10/2013
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DOI: 10.1002/eji.201343463
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Induced and thymus-derived Foxp3+ regulatory T cells share a common niche Yi-Ju Huang1 , Verena Haist2 , Wolfgang Baumg¨ artner2 , Lisa F¨ ohse3 , Immo Prinz3 , Sebastian Suerbaum4 , Stefan Floess∗1 and Jochen Huehn∗1 1
Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Department of Pathology, University of Veterinary Medicine, Hannover, Germany 3 Institute of Immunology, Hannover Medical School, Hannover, Germany 4 Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany Foxp3+ regulatory T (Treg) cells, which play a central role for the maintenance of immune homeostasis and self-tolerance, are known to be both generated in the thymus (thymusderived, tTreg cells) and in the periphery, where they are converted from conventional CD4+ T cells (induced Treg (iTreg) cells). Recent data suggest a division of labor between these two Treg-cell subsets since their combined action was shown to be essential for protection in inflammatory disease models. Here, using the transfer colitis model, we examined whether tTreg cells and iTreg cells fill different niches within the CD4+ T-cell compartment. When naive T cells were co-transferred with either pure tTreg cells or with a mixture of tTreg cells and iTreg cells, induction of Foxp3+ Treg cells from naive T cells was not hampered by preoccupation of the Treg-cell niche. Using neuropilin-1 (Nrp1) as a surface marker to separate tTreg cells and iTreg cells, we demonstrate that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and display comparable TCR repertoires. However, when transferred together Nrp1+ tTreg cells outcompeted Nrp1− iTreg cells and dominated the Treg-cell compartment. Taken together, our data suggest that tTreg cells and iTreg cells share a common peripheral niche.
Keywords: Colitis r iTreg cells
r
Neuropilin-1 r Treg cells
r
tTreg cells
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction Regulatory T (Treg) cells, expressing the lineage specification factor Foxp3, play a central role in immune tolerance to self and innocuous foreign antigens [1]. The majority of Foxp3+ Treg cells is generated during thymic development (thymus-derived, tTreg cells) and is believed to be selected for recognition of self-antigens.
Correspondence: Prof. Jochen Huehn e-mail: [email protected] C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In the periphery, the Treg-cell repertoire needs to be complemented by specificities directed against nonpathogenic foreign antigens, including commensal microbiota and food antigens, and this expansion of the Foxp3+ Treg-cell pool is achieved by peripheral conversion of conventional Foxp3− CD4+ T cells into Foxp3+ Treg cells (induced Treg (iTreg) cells) [2–5]. Recently, Helios, a member of the Ikaros transcription factor family, was
∗
These authors share senior co-authorship.
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suggested as a marker to distinguish between tTreg cells and iTreg cells [6, 7], however Helios has also been associated with T-cell activation and differentiation, and under certain conditions can be upregulated in iTreg cells [3, 8, 9]. More recently, the cell surface receptor neuropilin-1 (Nrp1), which was initially identified in a microarray-based approach as a Treg-cell-specific molecule [10], was reported to discriminate between Nrp1+ tTreg cells and Nrp1− iTreg cells particularly under steady-state conditions [11, 12]. Several studies have demonstrated that iTreg cells are capable of suppressing autoimmune responses to a similar degree as tTreg cells [2, 3, 13] and play a central role for the acquisition of mucosal as well as fetomaternal tolerance [14, 15]. Interestingly, full suppression in these autoimmune disease models was only achieved in the combined presence of both tTreg cells and iTreg cells [3, 13], and this complementary action was explained by the unique TCR repertoire of iTreg cells, which expanded the TCR diversity of the total Treg-cell pool [3, 4]. In the present study, we asked whether tTreg cells and iTreg cells occupy separate niches or whether they can supplement each other and fill a common Treg-cell niche. By performing adoptive transfer studies into lymphopenic recipient mice, we demonstrate that de novo induction of Foxp3+ Treg cells is not prevented by the presence of iTreg cells, that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and that tTreg cells dominate over iTreg cells when transferred under competitive conditions.
Results Preoccupation of the Treg-cell niche does not influence de novo generation of Foxp3+ Treg cells It is widely accepted that a division of labor exists between tTreg cells and iTreg cells and that both subsets are essential for the suppression of inflammatory immune responses in various tissues [2, 13, 15–17]. Nevertheless, it is only incompletely understood if de novo generation of iTreg cells is influenced by a preoccupation of the peripheral Treg niche. To address this question, we used the T-cell transfer colitis model, in which adoptive transfer of naive Foxp3− CD4+ T cells into immunodeficient recipient mice results in severe intestinal inflammation a few weeks after transfer, which can be prevented by co-transfer of Foxp3+ Treg cells [18]. Importantly, under these lymphopenic conditions, a significant fraction of transferred naive T cells converts into Foxp3+ iTreg cells in situ [19], and these iTreg cells were shown to play an important role for the full prevention of colitis [13]. Here, congenically marked naive Thy1.1+ T cells were adoptively transferred into SCID recipient mice either alone or co-transferred with a low (ratio 8:1) or a high (ratio 1:1) number of Thy1.1− Foxp3+ Treg cells. Five weeks after transfer, de novo generated iTreg cells could be detected among Thy1.1+ CD3+ CD4+ cells isolated from colonic lamina propria (cLP), mesenteric lymph node (mLN) and skin-draining, peripheral lymph node (pLN) (Fig. 1A and B), without observing a preferential iTreg-cell generation at mucosal sites as had been reported before [5, 19]. De novo generation of iTreg C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. De novo induction of Treg cells is independent of the presence of co-transferred Treg cells. Naive T cells (Thy1.1+ ) were adoptively transferred alone (1:0 ratio) or together with CD4+ Foxp3GFP+ Treg cells (Thy1.1− ) isolated from spleens and LN of DEREG mice into SCID recipients at indicated ratios. (A) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ -gated cLP lymphocytes isolated from recipient mice 5 weeks after transfer. Numbers indicate frequency of cells within quadrants. (B) The frequency of de novo induced Foxp3+ cells among total Thy1.1+ CD3+ CD4+ cells and (C) the frequency of total Foxp3+ Treg cells among CD3+ CD4+ cells in pLN, mLN, and cLP of indicated groups are shown as mean + SD of n = 5–6 mice from one experiment representative of two independent experiments. n.s.: not significant, *p < 0.05, **p < 0.01, Kruskal–Wallis and Mann–Whitney test.
cells not only occurred in SCID mice receiving naive T cells alone but also in mice that received naive T cells together with Treg cells (Fig. 1A). Strikingly, the frequency of de novo generated iTreg cells (Thy1.1+ Foxp3+ ) was largely independent of the presence of co-transferred Thy1.1− Treg cells and the ratio between naive T cells and Thy1.1− Treg cells (Fig. 1B). Comparable results were achieved when a higher cell number was used for adoptive transfer of naive T cells and Treg cells at a ratio of 8:1 (Supporting Information Fig. 1). The absolute number of de novo generated iTreg cells (Supporting Information Fig. 2A) and the frequency of total Foxp3+ Treg cells (Fig. 1C) was influenced by the number of initially co-transferred Treg cells, but no significant differences between total Treg-cell numbers between the different groups were observed (Supporting Information Fig. 2B). These findings can be best explained by the fact that co-transferred Treg cells not only affected total lymphocyte numbers (Supporting Information Fig. 2C), but also the strength of the inflammatory response within the colon (Supporting Information Fig. 3). Together, our results www.eji-journal.eu
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Figure 2. De novo induction of Foxp3+ Treg cells is not impaired in the presence of iTreg cells. Naive T cells (Thy1.1+ ) were adoptively transferred together with CD4+ Foxp3GFP+ Treg cells (Thy1.1− ) isolated from thymus (thymic-Treg), mLN (mLN-Treg) or pLN (pLN-Treg) of DEREG mice into SCID recipients at a ratio of 8:1. (A) Representative flow cytometry data showing Helios expression among CD4+ Foxp3GFP+ Treg cells isolated from thymus, mLN or pLN of DEREG mice. Numbers indicate frequency of Helios+ cells and shaded histograms represent isotype controls. (B) The frequency of Helios+ (black bar) and Helios− (white bar) cells among CD4+ Foxp3GFP+ cells from the indicated organs are shown as mean + SD of n = 3 from one experiment representative of three independent experiments performed. (C) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ cLP lymphocyte isolated from recipient mice 10 weeks after transfer. Numbers indicate frequency of cells within quadrants. (D) The frequency of de novo induced Foxp3+ cells among total Thy1.1+ CD3+ CD4+ cells and (E) the proportion of Thy1.1+ (dashed bars) and Thy1.1− (filled bars) Foxp3+ Treg cells among CD3+ CD4+ cells isolated from pLN, mLN, and cLP of indicated groups are shown as mean + SD of n = 6 from one experiment representative of two independent experiments performed. n.s., not significant, Kruskal–Wallis test.
show that de novo induction of Treg cells is not prevented but modulated by the preexistence of Foxp3+ Treg cells.
De novo induction of Foxp3+ Treg cells is not influenced by presence of iTreg cells Our finding that co-transfer of total Treg cells does not prevent de novo generation of iTreg cells did not answer the question if iTreg cells do occupy a unique niche that is different from the niche for tTreg cells and if the preexistence of iTreg cells influences de novo generation of Treg cells from naive T cells. To address this issue, Foxp3+ Treg cells were isolated from thymi, mLN, or pLN of Thy1.1− donor mice and co-transferred together with Thy1.1+ naive T cells into SCID recipient mice. Treg cells isolated from the thymus can be regarded as a largely pure tTreg-cell population, showing the highest frequency of Helios+ cells (Fig. 2A and B) and containing only scarce recirculating peripheral Treg cells [20]. In contrast, mLN- and pLN-derived Treg cells are known to contain a substantial fraction of iTreg cells, which are believed to reside mainly within the Helios− fraction (Fig. 2A and B). Ten weeks after transfer, histopathological analyses of colonic tissues revealed no significant differences in the strength of C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the inflammatory response between groups of mice receiving thymus-, mLN- or pLN-derived Treg cells (Supporting Information Fig. 4). Interestingly, frequencies and absolute numbers of de novo generated iTreg cells (Thy1.1+ Foxp3+ ) within pLN, mLN, and cLP were comparable between the different groups (Fig. 2C and D and Supporting Information Fig. 5A), suggesting that the preexistence of iTreg cells does not significantly hamper de novo generation of iTreg cells. Moreover, 10 weeks after transfer frequencies of total Treg cells as well as absolute numbers of total Treg cells and total lymphocytes were comparable within all organs analyzed between groups of mice receiving thymus-, mLN- or pLN-derived Treg cells (Fig. 2E and Supporting Information Fig. 5B and C), suggesting that the size of the total Treg-cell compartment in lymphopenic recipient mice is independent of the origin of transferred Treg cells. The results obtained so far suggested that iTreg cells do not occupy a unique niche, but rather share a common niche with tTreg cells. To exclude that the fraction of iTreg cells within mLN-Treg cells or pLN-Treg cells was too small to significantly influence de novo generation of iTreg cells from naive T cells, we next used the recently described cell surface marker Nrp1 [11, 12] to isolate iTreg cells (Nrp1− ) and tTreg cells (Nrp1+ ) from spleens of healthy Thy1.1− mice for co-transfer experiments.
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Figure 3. Co-transfer of Nrp1− iTreg cells does not affect de novo generation of iTreg cells and results in a completely filled Treg-cell niche. Naive T cells (Thy1.1+ ) were adoptively transferred together with Nrp1+ or Nrp1− CD4+ Foxp3GFP+ Treg-cell subsets (Thy1.1− ) isolated from spleens of DEREG mice into SCID recipients at a ratio of 8:1. (A) Representative flow cytometry data show Nrp1 versus Foxp3GFP expression on gated CD4+ splenocytes before sort (left) as well as sort purity and Helios expression within sorted Treg-cell subsets (right). Numbers indicate frequency of gated cells and shaded histograms represent isotype controls. (B) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ gated cLP lymphocyte isolated from recipient mice 10 weeks after transfer. Numbers indicate frequency of cells within quadrants (C) The stability of Foxp3 expression in transferred Thy1.1− Foxp3+ Treg cells (gated on total Thy1.1− CD3+ CD4+ cells), (D) the frequency of de novo induced Foxp3+ cells among total Thy1.1+ CD3+ CD4+ cells and (E) the proportion of Thy1.1+ (dashed bars) and Thy1.1− (filled bars) Foxp3+ Treg cells among CD3+ CD4+ cells in pLN, mLN, and cLP of indicated groups are shown as mean + SD of n = 11 pooled from two independent experiments. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Mann–Whitney test.
Sorted Foxp3+ Nrp1+ tTreg cells homogeneously expressed Helios and Foxp3+ Nrp1− iTreg cells were largely Helios− (Fig. 3A). Ten weeks after transfer, histopathological analyses of colonic tissues revealed no significant differences in the strength of the inflammatory response between groups of mice receiving Nrp1+ or Nrp1− Treg cells (Supporting Information Fig. 6). As expected from the results of a recent study introducing Nrp1 as a marker to discriminate between tTreg cells and iTreg cells [11, 12], Nrp1+ tTreg cells showed a rather stable Foxp3 expression, whereas Nrp1− iTreg cells had substantially lost Foxp3 expression in pLN, mLN, and cLP 10 weeks after transfer (Fig. 3B and C). We next determined the frequency of de novo generated iTreg cells (Thy1.1+ Foxp3+ ). Surprisingly, we observed a significantly higher iTreg generation in pLN, mLN, and cLP of mice being cotransferred with Nrp1− iTreg cells compared to mice receiving Nrp1+ tTreg cells (Fig. 3B and D), and a significantly higher absolute number of iTreg cells was found in the cLP of mice that had received Nrp1− iTreg cells when compared to mice that had received Nrp1+ tTreg cells (Supporting Information Fig. 7A). Importantly, frequencies of total Treg cells (Fig. 3E) as well as C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
absolute numbers of total Treg cells and total lymphocytes were not different between both groups of mice within all organs analyzed (Supporting Information Fig. 7 B and C), showing that both Nrp1− iTreg cells and Nrp1+ tTreg cells could uniformly fill the total Treg niche. Viewed as a whole, our data strongly argue against the existence of a unique iTreg-cell niche, but support the notion that iTreg cells rather share a common niche with tTreg cells in the periphery.
Nrp1+ tTreg cells outcompete Nrp1− iTreg cells to dominate the Treg-cell compartment High diversity of the TCR repertoire is of utmost importance for the homeostasis and function of Treg cells [21]. Our finding that Nrp1+ tTreg cells and Nrp1− iTreg cells are equally potent to uniformly fill the entire Treg-cell niche in lymphopenic mice suggested that both Treg-cell subsets displayed a comparable TCR repertoire. Thus, we analyzed the TCR repertoire in Nrp1+ and Nrp1− Treg cells isolated from spleens of Foxp3 reporter mice
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Figure 4. TCR repertoire of Nrp1+ tTreg and Nrp1− iTreg subsets. CD4+ T cells from spleens of Thy1.1+ Foxp3hCD2 mice were sorted into Nrp1+ Treg cells (black), Nrp1− Treg cells (white), and Foxp3− cells (gray) and their Tcra repertoire was analyzed by next-generation sequencing. The Jα usage of functionally rearranged TRAV12 (Vα8) family sequences for each population is shown as mean + SD of four individual samples from one experiment.
(Fig. 4). Analysis of the TRAV12 (Vα8) family, which constitutes approximately 10% of all rearranged TCR-α chains in murine CD4+ T cells, revealed an overall similar J usage pattern between both Treg-cell subsets and even Foxp3− CD4+ T cells (Fig. 4). A comparable degree of sequence overlap among all three populations was also evident when comparing similarities by Morisita–Horn indices of the respective CDR3 regions (Supporting Information Fig. 8A and B). This indicates that Nrp1+ and Nrp1− Treg cells used in the former experiment (Fig. 3) were equipped with similar TCR repertoires. Having shown that Nrp1+ tTreg cells and Nrp1− iTreg cells can both uniformly fill the entire Treg-cell niche and display similar TCR repertoires, we next asked how these two Tregcell subsets behave under competitive conditions. To this end, we adoptively transferred Thy1.1+ naive T cells together with 4:1 or 1:4 mixtures of Nrp1+ tTreg cells and Nrp1− iTreg cells into Rag2-deficient mice (Fig. 5A). The Treg-cell subsets were Thy1.1− and either CD45.1+ or CD45.2+ to allow definite identification. A swap of donor mice was included in the experimental setup to avoid a strain-driven bias. Eight weeks after adoptive transfer, histopathological analyses of colonic tissues revealed no significant differences in the strength of the inflammatory response between groups of mice receiving 4:1 or 1:4 mixtures of Nrp1+ tTreg cells and Nrp1− iTreg cells (Supporting Information Fig. 9A). Furthermore, frequencies and absolute numbers of de novo generated iTreg cells (Thy1.1+ Foxp3+ ) within pLN, mLN, and cLP were comparable between the two groups (Supporting Information Fig. 9B and C). Strikingly, when we analyzed the composition of the Thy1.1− Foxp3+ Tregcell compartment, we observed that the tTreg-cell progeny dominated the Treg-cell compartment in both groups receiving a mixture of Treg cells (Fig. 5A and B). Absolute numbers of Thy1.1− Foxp3+ cells and total lymphocytes did not show any significant differences between the two groups at different locations (Supporting Information Fig. 9D and E). In summary, these results demonstrate that tTreg cells can outcompete iTreg cells if transferred together and dominate the total Treg-cell niche at the endpoint of the experiment.
Discussion The peripheral pool of Foxp3+ Treg cells is a heterogeneous population of tTreg cells and iTreg cells. In the present study, we asked whether tTreg cells and iTreg cells occupy separate niches C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. Nrp1+ tTreg cells outcompete Nrp1− iTreg cells and dominate the Treg-cell compartment. Naive T cells (Thy1.1+ ) were adoptively transferred together with a mixture of Nrp1+ and Nrp1− CD4+ Foxp3+ Treg-cell subsets (Thy1.1− ) into RAG2−/− recipients at a ratio of 8:1. The Treg-cell mixture consisted of Nrp1+ and Nrp1− Treg cells isolated from spleens of FIR (CD45.2+ ) or Foxp3hCD2 Rag1GFP (CD45.2− ) mice at a ratio of 4:1 or 1:4. (A) Representative flow cytometry data showing Foxp3 protein versus Thy1.1 expression in CD3+ CD4+ gated cells (left) and CD45.2 expression in CD3+ CD4+ Thy1.1− Foxp3+ gated cells (right) isolated from mLN of recipient mice 8 weeks after transfer. Numbers indicate frequency of cells within quadrants or gated cells. (B) The frequency of Nrp1+ progeny among CD3+ CD4+ Thy1.1− Foxp3+ cells in pLN, mLN, and cLP of indicated groups is shown as mean ± SD of n = 7–8 pooled from two independent experiments.
or whether they can supplement each other and fill a common Treg-cell niche. By performing adoptive transfer studies into lymphopenic recipient mice, we could demonstrate that de novo induction of Foxp3+ Treg cells is not prevented by the presence of iTreg cells, that tTreg cells and iTreg cells share a common Tregcell niche in the periphery and that tTreg cells dominate over iTreg cells if transferred under competitive conditions. Adoptive transfer of naive Foxp3− CD4+ T cells into lymphopenic recipient mice is known to result in severe intestinal inflammation a few weeks after transfer [18]. Although previous in vitro studies have reported that de novo induction of Foxp3+ Treg cells is negatively influenced by inflammatory cytokines [22–24], in the present study, we observed efficient iTreg-cell generation even in mice that did not receive any co-transferred Treg cells and www.eji-journal.eu
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that developed severe colitis 5 weeks after T-cell transfer. A scenario in which de novo induction of Foxp3+ Treg cells precedes the development of colitis in the recipient mice can best explain this discrepancy. Indeed, an impaired de novo induction of Foxp3+ Treg cells had been reported when sublethally irradiated mice, which immediately suffer from irradiation-induced inflammation, were used as recipients [25]. The functional properties of iTreg cells in the transfer colitis model are currently controversially discussed. Although a recently published paper showed that early development of iTreg cells could alleviate intestinal inflammation [26], in the present study, we observed no correlation of the frequency of iTreg cells with the colitis score. In line with our data, Izcue et al. reported a similar degree of colitis regardless whether the lymphopenic recipient mice did receive Foxp3-deficient or -sufficient naive T cells [27]. Despite these discrepancies, all studies agree on the fact that the local inflammatory milieu and the severity of the disease critically influence the functional properties of iTreg cells. Previous studies have reported a high degree of similarity between iTreg cells and tTreg cells as they show both similar gene expression profiles [3, 14] as well as comparable epigenetic signatures [28]. However, differences in the TCR repertoire seem to be dependent on the location of the Treg cells, especially the TCR repertoire of colonic Treg cells might be microbiota driven and therefore different from others [4]. In the present study, Nrp1+ tTreg cells and Nrp1− iTreg cells used for adoptive transfer experiments were isolated from the spleen, a place that is known to contain Treg cells with a less diverse TCR repertoire [29]. Accordingly, we did observe comparable TCR repertoires in Nrp1+ tTreg cells and Nrp1− iTreg cells, but presumably those TCR repertoires develop differently after transfer into lymphopenic hosts as it was recently published [3]. Nrp1 has been recently reported to discriminate between tTreg cells and iTreg cells, particularly under steady-state conditions [11, 12]. Although Nrp1 expression was shown to be influenced by TGF-β and inflammatory environments [3,11], this cell surface marker correlates to a high degree with the intracellular tTregcell marker Helios when healthy SPF-housed mice were taken as donors. After transfer of Nrp1+ tTreg cells or Nrp1− iTreg cells together with naive T cells into lymphopenic recipients, we found that both Treg-cell subsets could uniformly fill the entire Treg-cell niche. This is particularly worth mentioning in case of adoptive transfer of Nrp1− iTreg cells together with naive T cells since under these conditions tTreg cells are completely lacking in the recipient mice. Importantly, we here observed an even higher iTreg generation compared to mice receiving Nrp1+ tTreg cells, but this somewhat unexpected observation can be best explained by differences in the stability of Foxp3 expression between Nrp1− iTreg cells and Nrp1+ tTreg cells after transfer into immunodeficient mice. Indeed, Nrp1+ tTreg cells were recently reported to display a more complete demethylation of the foxp3 locus compared to Nrp1− iTreg cells [11], a phenotype that is known to correlate with stability of Foxp3 expression [30]. These data strongly argue against the existence of unique subniches for iTreg cells and tTreg C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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cells and is somewhat contrary to recently published data analyzing a mouse mutant that lacks the conserved noncoding sequence 1 (CNS1) of the foxp3 locus, a TGF-β − responsive element that has been suggested to play an important role for the generation of iTreg cells [14]. Co-transfer of Foxp3+ Treg cells from CNS1deficient mice together with naive T cells into lymphopenic recipient mice resulted in a higher de novo generation of iTreg cells when compared with that after co-transfers using Foxp3+ Treg cells from WT mice [14]. However, CNS1−/− mice still contained Nrp1− Foxp3+ Treg cells [11], suggesting that a significant fraction of iTreg cells can be generated independently of TGF-β. It thus still remains to be clarified whether TGF-β-dependent and -independent iTreg cells occupy unique subniches within the total Treg-cell compartment. The ability of tTreg cells and iTreg cells to uniformly fill the entire Treg niche raised the question about the relationship between these subsets. In view of the fact that there is a high degree of similarity in the gene expression profiles [3, 14], epigenetic signatures [28] and TCR repertoires (present study), it was somewhat unexpected to see a clear dominance of tTreg cells above iTreg cells in competitive co-transfer experiments, even when the majority of the transferred cells originate from Nrp1− iTreg cells. This finding can be explained by a different migratory behavior of Nrp1+ tTreg cells and Nrp1− iTreg cells, as recently reported in a tumor model using mice that lack Nrp1 selectively on CD4+ T cells [31]. Alternatively, ligation of Nrp1 on the surface of tTreg cells by its ligand semaphorin-4a can modulate Akt-mTor signaling, which finally promotes Treg-cell stability by enhancing quiescence, increasing expression of survival factors, slightly increasing proliferation, and concomitantly repressing terminal differentiation [32]. Together, these changes can explain our observation that Nrp1+ tTreg cells outcompete Nrp1− iTreg cells and dominate the entire Treg-cell niche when transferred together. In conclusion, the findings of the present study suggest that peripheral de novo induction of Foxp3+ Treg cells from naive T cells in lymphopenic models is not hampered by the preexistence of iTreg cells. An empty Treg-cell niche can be entirely filled by either tTreg cells or iTreg cells, providing strong arguments for a common Treg-cell niche in the periphery.
Materials and methods Mice DEREG mice [33], congenic BALB/c × Thy1.1+ mice, congenic BALB/c × Thy1.1+ Foxp3hCD2 mice ([34], kindly provided by Shohei Hori, Yokohama, Japan), Rag2-deficient C57BL/6 mice (C57BL/6 background), Foxp3IRES-RFP mice ([25], C57BL/6 background, kindly provided by Richard Flavell, New Haven, CT, USA), CD45.1+ Foxp3hCD2 Rag1GFP mice ([20], C57BL/6 background, kindly provided by Shohei Hori, Yokohama, Japan) and congenic www.eji-journal.eu
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C57BL/6J Thy1.1+ mice were bred under specific pathogen-free conditions at the animal facility of the Helmholtz Centre for Infection Research (Braunschweig, Germany). SCID mice and Thy1.2+ BALB/c mice were purchased from Charles River Germany. Animal care and all procedures were performed in accordance with institutional, state, and federal guidelines.
Nrp1− iTreg cells (tTreg:iTreg ratio 4:1) isolated from spleens of Foxp3IRES-RFP or CD45.1+ Foxp3hCD2 Rag1GFP . In one experiment, 9 × 105 naive T cells were adoptively transferred together with 1.125 × 105 CD4+ Foxp3hCD2+ Treg cells into SCID recipient mice. Body weight of recipients was monitored regularly, and mice were sacrificed when losing 20% of highest body weight.
Antibodies, staining, and flow cytometry
Purification of lamina propria lymphocytes
The following antibodies were purchased from eBioscience: antiCD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), antiCD25 (PC61.5), anti-CD11c (N418), anti-CD90.1 (HIS51), antiCD90.2 (53–2.1), anti-CD45R (RA3–6B2), anti-CD103 (2E7), anti-GITR (DTA-1), anti-Foxp3 (FJK-16s). Anti-hCD2 (RPA-2.10), anti-CD45.2 (104), anti-CD45RB (C363.16A), and anti-Helios (22F6) were purchased from Biolegend and biotinylated antiNrp1 (polyclonal) was purchased from R&D. Cells were stained with LIVE/DEAD Fixable Dead Cell stains (Invitrogen), followed by fluorochrome-conjugated surface marker staining. Cells were fixed, permeabilized, and stained intracellularly for Helios and Foxp3 using the Foxp3 staining set (eBioscience). Samples were acquired with an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Lymphocytes from large intestines of recipient mice were prepared as previously described [7] with minor modifications. Briefly, tissues were digested in Liberase Blendzyme (0.1 U/mL, Roche) and DNase I (0.1 mg/mL, Sigma) containing RPMI 1640 (Gibco) buffer for 45 min at 37◦ C followed by a 40%:70% Percoll gradient (Easycoll, Biochrom AG). Lamina propria lymphocytes were obtained from the interface of the gradient and resuspended in PBS buffer for further staining.
Cell sorting and adoptive transfer Naive CD4+ T cells were isolated from spleen and LN singlecell suspensions of 6–8 week old Thy1.1 mice by first enriching CD4+ cells using anti-CD4 microbeads and the autoMACS magnetic separation system (Miltenyi Biotec). Enriched CD4+ cells were stained with anti-CD4, anti-CD45RB, anti-GITR antibodies and the exclusion marker antibodies anti-CD8, antiCD11c, anti-B220, anti-CD25, and anti-CD103, and sorted into naive CD4+ CD45RBhigh GITRlow T cells, negative for the exclusion markers, by fluorescence-activated cell sorting (FACS) (FACSAria, BD Biosciences). For the isolation of organ-specific CD4+ Foxp3GFP+ Treg cells, CD4+ cells were first enriched from pLN, mLN, and thymic single-cell suspensions of 6–8 week old DEREG mice by anti-CD4 microbeads (pLN and mLN) or CD8Dynabeads (Invitrogen) to deplete CD8+ thymocytes. Enriched CD4+ T cells were stained with anti-CD4 and sorted into CD4+ Foxp3GFP+ Treg cells by FACS. For the isolation of Nrp1+ and Nrp1− CD4+ Foxp3+ Treg subsets, CD4-enriched splenocytes from DEREG, Foxp3IRES-RFP or CD45.1+ Foxp3hCD2 Rag1GFP mice were stained with anti-CD4, anti-Nrp1 and anti-hCD2, and sorted by FACS. Purity of sorted cells generally was ∼ 99%. 1.8 × 105 naive T cells were adoptively transferred by i.p. injection either alone (1:0 ratio) or together with 1.8 × 105 (1:1 ratio) or 2.25 × 104 (8:1 ratio) sorted CD4+ Foxp3GFP+ Treg cells derived from DEREG mice or a congenically marked Treg-cell mixture into SCID or Rag2−/− recipient mice. The Treg mixture consisted of either 4.5 × 103 Nrp1+ tTreg cells plus 1.8 × 104 Nrp1− iTreg cells (tTreg:iTreg ratio 1:4) or 1.8 × 104 Nrp1+ tTreg cells plus 4.5 × 103 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
TCR amplification and high-throughput sequencing Total RNA was isolated using the RNeasy Kit (Qiagen) from 70 000 FACS-sorted CD4+ Foxp3+ Nrp1+ , CD4+ Foxp3+ Nrp1− , or CD4+ Foxp3− cells, from four individually analyzed donors. cDNA templates were synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche) according to manufacturer’s recommendation. Amplicon libraries of rearranged TRAV12 (Vα8 family) CDR3 regions for the Genome Sequencer FLX System (454 sequencing, Roche) were generated as described before [21]. Amplicons were purified by agarose gel electrophoresis and QIAquick Gel Extraction Kit (Qiagen), and quantified by QuantiTTM dsDNA HS Assay Kit (Invitrogen). Sequencing reactions were performed by ultra-deep 454 pyrosequencing on the Genome Sequencer FLX system (Roche Applied Sciences) according to manufacturer’s recommendation. Productive rearrangements, J usage, and CDR3α regions were defined by comparing nucleotide sequences to the reference sequences from IMGTs, the international ImMunoGeneTics information systems (http://www.imgt.org; IMGT/HighV-QUEST) [35, 36].
Histology Two pieces from the colon of each recipient mouse were dissected, fixed in formalin, and embedded in paraffin. Tissue slices were stained with hematoxylin and eosin. Blinded sections of colon pieces were examined and classified into a five-level score [37].
Statistics Values generated by adoptive transfer experiments were analyzed by GraphPad Prism (GraphPad Software) with mean ± SD and calculated by Mann–Whitney test or Kruskal–Wallis test. P www.eji-journal.eu
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values < 0.05 were considered as statistically significant; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. For the analysis of the TCR repertoire, the similarity between two sequenced TCR repertoires was statistically measured by the Morisita–Horn index [38]. This index ranges between 0 (completely dissimilar) and 1 (identical) and is comparatively resistant to sample size. To compare all samples the cut off was set to 6700 productive sequences per sample.
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T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression. PLoS One 2011. 6: e20731. 10 Bruder, D., Probst-Kepper, M., Westendorf, A. M., Geffers, R., Beissert, S., Loser, K., von Boehmer, H. et al., Neuropilin-1: a surface marker of regulatory T cells. Eur. J. Immunol. 2004. 34: 623–630. 11 Weiss, J. M., Bilate, A. M., Gobert, M., Ding, Y., Curotto de Lafaille, M. A., Parkhust, C. N., Xiong, H. et al., Neuropilin-1 is expressed on thymusderived natural regulatory T cells, but not mucosa-generated induced Foxp3+ Treg cells. J. Exp. Med. 2012. 209: 1723–1742. 12 Yadav, M., Louvet, C., Davini, D., Gardner, J. M., Martinez-Llordella, M., Bailey-Bucktrout, S., Anthony, B. A. et al., Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J. Exp. Med. 2012. 209: 1713–1722. 13 Haribhai, D., Lin, W., Edwards, B., Ziegelbauer, J., Salzman, N. H., Carl-
Acknowledgements: We thank Lothar Gr¨ obe, Maria Ebel, Beate Pietzsch, and Sabrina Woltemate for excellent technical assistance. We thank Shohei Hori (Yokohama, Japan) and Richard Flavell (New Haven, CN) for providing Foxp3 reporter mice. This work was supported by a grant from the German Research Foundation (SFB621), the Hannover Biomedical Research School (HBRS) and the Center for Infection Biology (ZIB).
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Conflict of interest: The authors declare no financial or commercial conflict of interest.
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C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Full correspondence: Prof. Jochen Huehn, Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany Fax: +49-531-6181-3399 e-mail: [email protected] Received: 19/2/2013 Revised: 2/9/2013 Accepted: 1/10/2013 Accepted article online: 8/10/2013
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