DOI: 10.1002/eji.201444532
Eur. J. Immunol. 2015. 45: 167–179
Immunomodulation
Foxp3+ regulatory T cells ensure B lymphopoiesis by inhibiting the granulopoietic activity of effector T cells in mouse bone marrow Sunghoon Kim ∗1 , Kyungsoo Park ∗2 , Jinwook Choi2 , Eunkyeong Jang3 , Doo-Jin Paik3 , Rho H. Seong ∗∗2 and Jeehee Youn ∗∗1,3 1
Department of Biomedical Sciences, Hanyang University Graduate School, Seoul, Korea Department of Biological Sciences, Research Center for Functional Cellulomics, Seoul National University, Seoul, Korea 3 Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul, Korea 2
Foxp3+ Treg cells are crucial for maintaining T-cell homeostasis, but their role in B-cell homeostasis remains unclear. Here, we found that Foxp3 mutant scurfy mice had fewer B-lineage cells and progenitors, including common lymphoid progenitors and lymphoidprimed multipotent progenitors, but higher myeloid-lineage cell numbers in BM compared with WT littermates. Homeostasis within the HSC compartment was also compromised with apparent expansion of long- and short-term HSCs. This abnormality was due to the lack of Treg cells, but not to the Treg-cell extrinsic functions of Foxp3 or cellautonomous defects. Among cytokines enriched in the BM of scurfy mice, IFN-γ affected only B lymphopoiesis, but GM-CSF, TNF, and IL-6 collectively promoted granulopoiesis at the expense of B lymphopoiesis. Neutralization of these three cytokines reversed the hematopoietic defects on early B-cell progenitors in scurfy mice. Treg cells ensured B lymphopoiesis by reducing the production of these cytokines by effector T cells, but not by directly affecting B lymphopoiesis. These results suggest that Treg cells occupy an important niche in the BM to protect B-lineage progenitor cells from excessive exposure to a lymphopoiesis-regulating milieu.
Keywords: B lymphopoiesis r Foxp3
r
Granulopoietins r Hematopoiesis r Regulatory T cells
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction CD4+ Foxp3+ Treg cells are essential for maintaining immune homeostasis, as evidenced by the severe autoimmune lymphoproliferative disease observed in Treg-cell-deficient mice and humans [1, 2]. Treg cells were initially identified in the secondary lymphoid tissues where they suppress the initial priming and
Correspondence: Prof. Jeehee Youn e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
effector differentiation of autoreactive T cells [3]. They function by directly suppressing T cells and/or interacting with DCs to limit their ability to prime T cells effectively [4, 5]. In addition to their constitutive presence in secondary lymphoid tissues, Treg cells have been found in several nonlymphoid sites, such as mucosal tissue, skin, liver, and lung, during inflammation as well as in steady-state conditions [6–10]. These Treg cells appear to regulate
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These authors contributed equally to this work. These senior authors contributed equally to this work.
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tissue-tropic autoimmunity and inflammation, and alter pathogen clearance during peripheral infection. A diverse array of adhesion molecules and chemokine receptors that are expressed on Treg cells seem to license them to migrate to peripheral tissues. A fraction of Treg cells have been shown to express the BM homing receptor CXCR4 [11], suggesting the existence of a niche for Treg cells in the BM. Indeed, there are studies showing that healthy human BM contains a substantial number of Treg cells that traffic via CXCL12/CXCR4 signaling [12]. However, little is known of the role and significance of BM-residing Treg cells in the context of BM homeostasis. BM harbors a variety of hematopoietic lineage cells, including lymphoid and myeloid lineage cells [13–15]. All hematopoietic cells are derived from pluripotent HSCs. Long-term (LT)-HSCs lack all lineage markers (Lin− ) but express high levels of Sca-1 and c-Kit on their surface and give rise sequentially to short-term (ST)-HSCs and multipotent progenitors (MPPs), both of which are characterized by a loss of self-renewal capacity. MPPs are the most primitive progenitors, bifurcating into distinct lineage fates, namely common myeloid progenitors (CMPs) and lymphoidprimed multipotent progenitors (LMPPs). While CMPs give rise to granulocyte-monocyte progenitors (GMPs) and megakaryocyteerythrocyte progenitors (MEPs), LMPPs preferentially differentiate into common lymphoid progenitors (CLPs), a population of early lymphoid progenitors that has a reduced level of c-Kit and displays surface IL-7 receptor α chain. CLPs undergo a cascade of highly ordered steps until immature B cells emerge in the BM. After exit from the BM, immature B cells continue their maturation in the secondary lymphoid tissues to form the peripheral immune repertoire. Hematopoiesis is normally well controlled in order to replenish blood cells at a constant rate, such that the balance of lymphoid and myeloid cells is maintained. However, the output of particular blood cell types can be altered under certain conditions. For example, acute infections or acute allergic responses elevate the production of granulocytes at the expense of B lymphopoiesis, a phenomenon known as “emergency granulopoiesis” [16, 17]. This seems to be at least in part mediated by proinflammatory cytokines such as GM-CSF, TNF, and IL-6, since they are known to shift the developmental pathway of BM progenitors toward granulopoiesis [15, 18–20]. Whether the activity of Foxp3+ Treg cells also influences hematopoiesis in the BM remains a matter of debate, since contradictory roles of Treg cells in hematopoiesis have been reported, specifically in B-cell development in the BM [21–23]. Although it was reported that Treg cells can directly inhibit in vitro differentiation of myeloid progenitor cells in a contact-dependent manner, and this effect was corroborated in vivo when Treg cells were cotransferred with BM cells into immunocompromised mice [24], it was not clear whether this mechanism also applies to lymphoid progenitors. Moreover, the demonstration that administration of Treg cells could not rescue the defective B-cell development in scurfy mice [25] implies that the B lymphopenia in scurfy BM is independent of the deficiency in Treg cells. Therefore, although most, but not all, previous studies detected a defect in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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B lymphopoiesis in scurfy BM, the underlying mechanism remained to be elucidated. Here, we analyzed all the aspects of BM hematopoiesis, including B lymphopoiesis, in a viable line of scurfy mice and revealed the mechanism by which Treg cells regulate the composition of BM cell populations. Our in vivo and in vitro data, taken together, suggest that effector T (Teff) cells infiltrating the BM perturb normal B lymphopoiesis by producing diverse hematopoiesis-regulating cytokines, and that Treg cells present in the BM protect B-lineage progenitor cells from this activity of Teff cells but do not directly regulate the activity of hematopoietic progenitor cells.
Results Perturbed hematopoiesis in the BM of Foxp3-deficient mice Scurfy mice harboring a loss-of-function mutation of Foxp3 do not develop Foxp3+ Treg cells, and so they can be used to investigate the role of Treg cells in vivo. However, scurfy mice on the C57BL/6 background (referred as to B6sf) usually succumb to extremely severe inflammatory symptoms at about 4 weeks of age, so there are practical difficulties associated with using them. To overcome these, we generated male KRN TCR transgenic C57BL/6 mice bearing a scurfy allele on the X chromosome (referred to as K/Bsf). As expected, K/Bsf mice exhibited much more modest symptoms of inflammation, and about 80% survived until at least 7 weeks of age by which time all our experiments were completed (data not shown). We first determined whether the absence of Foxp3 altered BM hematopoiesis in the K/Bsf mice. BM from K/Bsf mice contained significantly more Gr-1+ Mac-1+ myeloid cells, fewer B220+ B-lineage cells, and fewer TER-119+ erythroid cells than BM from their transgenic nonscurfy littermates (referred to as K/B hereafter; Fig. 1A). We further traced the B-lineage population through all cell stages and found that both percentages and cell numbers of pro-B (B220lo IgM− CD43+ ), pre-B (B220lo IgM− CD43− ), immature B (B220lo IgM+ ) and mature/recirculating B (B220hi IgM+ ) cell populations were lower in K/Bsf BM than in K/B BM (Fig. 1B). Moreover, the ratio of pro-B cells to pre-B cells was higher in K/Bsf BM. Consistent with this observation, the difference of cell numbers between the two strains was largest at the pre-B-cell stage. When we subdivided the B220lo IgM− CD43+ cells into fractions A, B, and C, the frequency and number of cells in fraction A were greater than those in fractions B and C in K/Bsf BM, whereas in K/B BM the cell numbers in all three fractions were similar (Fig. 1C). Next, we examined whether the B lymphopenia observed in K/Bsf BM stemmed from impaired development of earlier progenitors of the B-lineage cells. The number of CLPs (Lin− c-Kitlo Sca-1lo IL-7R+ ) was found to decrease dramatically in K/Bsf BM (Fig. 1D). LMPPs (Lin− Sca-1+ c-Kit+ Flt3+ CD34+ ) were also lower, albeit not as low as CLPs, while LT-HSCs (Lin− Sca-1+ c-Kit+ Flt3− CD34− ) and ST-HSCs (Lin− Sca-1+ c-Kit+ Flt3− CD34+ ) www.eji-journal.eu
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Figure 1. Normal hematopoiesis is perturbed in the BM of K/Bsf mice. BM cells from K/B () and K/Bsf () mice were stained with Abs to lineagespecific markers and analyzed by flow cytometry. (A) Myeloid lineages (Mac-1+ Gr-1+ ), erythroid cells (TER 119+ ), and B-lineage cells (B220+ ) gated on whole live cells. (B) Pro-B plus pre-B cells (B220lo IgM− ), immature B cells (B220lo IgM+ ), and mature B cells (B220hi IgM+ ) within a live cell gate (top plots). Pro-B cells (B220+ CD43+ ) and pre-B cells (B220+ CD43− ) within a B220lo IgM− gate (middle plots). (C) Pre–pro B cells (fraction A, HSA− BP-1− ), early pro-B cells (fraction B, HSA+ BP-1− ) and late pro-B cells (fraction C, HSA+ BP-1+ ) gated on B220+ CD43+ IgM− cells. (D) CLP population (Lin− Sca-1low c-Kitlow ) gated on Lin− IL-7Rα+ cells. (E) LT-HSC (Lin− Sca-1+ c-Kit+ Flt3− CD34− ), ST-HSC (Lin− Sca-1+ c-Kit+ Flt3− CD34+ ), and LMPP (Lin− Sca1+ c-Kit+ Flt3+ CD34+ ) gated on Lin− Sca-1+ c-Kit+ cells. (F) CMP (Lin− Sca-1− c-Kit+ CD34+ CD16/32− ), GMP (Lin− Sca-1− c-Kit+ CD34+ CD16/32+ ), and MEP (Lin− Sca-1− c-Kit+ CD34− CD16/32− ) populations gated on Lin− Sca-1− c-Kit+ cells. (A–F) Representative FACS profiles and mean + SEM cell numbers (n = 6 mice/group) pooled from four independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t-test.
were increased (Fig. 1E). These results indicate that the impairment of B-cell development in Foxp3-deficient mice starts with developmental arrest at the most primitive progenitors, namely LT-HSCs and ST-HSCs. In another set of staining experiments to detect the alternative fate of ST-HSC differentiation, CMPs were found to be about 2.4-fold less frequent in K/Bsf BM than in K/B BM, indicating that the Foxp3 deficiency-dependent arrest at the stage of ST-HSCs also blocks the emergence of CMPs, as well as LMPPs (Fig. 1F). Interestingly, despite this reduction, there were more GMPs in K/Bsf BM than in K/B BM, while the opposite was true for MEPs. We confirmed that there were no significant differences between K/Bsf and B6sf mice in terms of the cellular profiles of primary and secondary immune compartments. Moreover, all the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
aspects of K/Bsf BM mentioned above were precisely mirrored in B6sf BM, indicating that expression of the KRN transgene did not affect hematopoiesis in the scurfy mice (Supporting Information Fig. 1).
K/Bsf BM cells impaired hematopoiesis is due to Treg-cell deficiency, but is independent of Foxp3 We examined the mechanism by which hematopoiesis, in particular B lymphopoiesis, is impaired in the K/Bsf mice. Because previous studies had demonstrated Foxp3 expression in non-Treg cells, such as BM-derived mesenchymal stromal cells, thymic stromal cells, and mammary cells [26–28], we first needed to address www.eji-journal.eu
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Figure 2. Developmental profiles of K/Bsf BM cells in vitro and in BM chimeric mice. (A) LSK cells purified from K/B and K/Bsf mice were cultured on OP-9 stromal cells in the presence of IL-7, SCF, and Flt3L for 12 days and analyzed by flow cytometry, gated on whole live cells. Representative FACS profiles and percentages of each cell population derived from K/B and K/Bsf LSK cells are shown. (B–F) T-cell-depleted BM cells from CD45.1+ C57BL/6 mice, T-cell-depleted BM cells from CD45.2+ K/Bsf mice, or a mixture of both BM cells were transferred into sublethally irradiated RAG2−/− recipients. Six weeks after transplantation, B-lineage populations in the recipient BM were examined by flow cytometry. Percentages of B-lineage cells among (B and C) donor-derived whole BM cells, (D) donor-derived B220lo IgM− BM cells, and (E) donor-derived B220lo IgM− CD43+ BM cells are shown. (F) Total cell numbers in the recipient BM are shown. (A–F) Data are presented as mean + SEM (n = 3 mice/group) and are representative of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001; Student’s t-test.
the question of whether the impairment could be attributed to the Treg-cell-extrinsic function of Foxp3. To this end, LSK cells (Lin− Sca-1+ c-Kit+ ) purified from K/Bsf mice and K/B mice were cultured under B-cell differentiation conditions for 12 days, and the phenotypes of the resulting cells were analyzed by flow cytometry. LSK cells from K/Bsf mice formed B220+ CD19− pre–pro B cells and the more mature B220+ CD19+ B cells to an extent similar to that observed in the K/B mice (Fig. 2A). Thus, LSK cells from the K/Bsf mice seemed to be unaffected in their potential to give rise to B cells, and the abnormal B lymphopoiesis might, therefore, result from a Foxp3 function extrinsic to the hematopoietic progenitor cells. However, the normal B lymphopoiesis observed in the K/Bsf BM cells in vitro was not observed in mice that received these same C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cells by adoptive transfer: K/Bsf BM cells transplanted into sublethally irradiated RAG2−/− mice failed to give rise to B-cell subsets, unlike Foxp3-intact congenic CD45.1+ BM cells (Fig. 2B–F). To see whether this defect in B-cell development could be corrected by intact BM cells, we generated mixed BM chimeras by transplanting a mixture of congenic CD45.1+ Foxp3+/+ BM cells (50%) with CD45.2+ K/Bsf BM cells (50%) into irradiated RAG2−/− recipients. In the resulting chimeras, the CD45.2+ K/Bsf BM cells gave rise to all the hematopoietic cell populations including B-lineage cells. This shows that the abnormal B lymphopoiesis in K/Bsf mice is not due to any intrinsic defect in the hematopoietic progenitors, since they give rise to B cells in the presence of some “element” derived from the control BM progenitors.
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Figure 3. Treg cells repair abnormal B lymphopoiesis of BM cells from K/Bsf mice. (A) RAG2−/− mice were infused with T-cell-depleted K/Bsf BM cells alone or together with Treg cells purified from CD45.1+ C57BL/6 mice and assayed 6 weeks later by flow cytometry. Data are shown as mean + SEM of four samples pooled from two independent experiments. (B) CD4+ CD25hi or CD4+ CD25− cells purified from CD45.1+ C57BL/6 mice were injected into K/Bsf mice. Three weeks later, the BM cells of recipient mice were examined by flow cytometry. Data are shown as mean + SEM of five samples and are pooled from three independent experiments. *p < 0.05, **p < 0.01; Student’s t-test.
To determine whether this element was in fact the population of Treg cells, we generated mixed BM chimeras by transferring a mixture of CD45.2+ K/Bsf BM cells with CD45.1+ Treg cells into irradiated RAG2−/− recipients, and found that the defects in B-lineage cell formation of the K/Bsf donors were substantially relieved (Fig. 3A). Thus, Treg cells seemed to be sufficient to ensure that K/Bsf donor cells formed B-lineage cells normally.
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We confirmed this finding by reconstituting K/Bsf mice with Treg cells. The mice were infused with either CD4+ CD25hi cells (presumed to be a Treg-cell population) or CD4+ CD25− cells purified from CD45.1+ C57BL/6 mice. Infusion with CD4+ CD25hi cells greatly reduced all the aberrant phenotypes of K/Bsf BM (Fig. 3B). Taken together, our data indicate that the abnormal hematopoiesis observed in K/Bsf BM stems from the absence of Treg cells rather than from a Treg-cell-independent function of Foxp3.
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cells on B lymphopoiesis was fully inhibited by the addition of Treg cells. Taken together, these results demonstrate that Treg cells protect B lymphopoiesis from the suppressive activity of soluble factors derived from Teff cells.
The cytokine milieu of K/Bsf BM is different from that of normal BM
Figure 4. Treg cells ensure B lymphopoiesis by controlling the suppressive activity of Teff cells. LSK cells purified from CD45.1+ C57BL/6 mice were cultured in the presence or absence of preactivated Treg cells or/and Teff cells purified from Foxp3gfp reporter mice. In some experiments, Teff cells were replaced by the supernatants from cultures of activated Teff cells (Teff sup) or Teff cells were added into transwells (tw). After 12 days of culture, differentiated B cells (B220+ CD19+ ) were examined by flow cytometry, gated on whole live cells. (A) FACS profiles are representative of three independent experiments. (B) Data are shown as mean + SEM percentages of B220+ CD19+ cells and are pooled from three independent experiments. ***p < 0.001; Student’s t-test. NS: not significant.
Treg cells protect B lymphopoiesis by controlling the suppressive activity of Teff cells Our finding that the scurfy-associated perturbation of B lymphopoiesis was due to Treg-cell-autonomous defects in Foxp3 function suggests that Treg cells play a crucial role in B lymphopoiesis in the BM. Whether Treg cells directly control B lymphopoiesis was assessed by culturing LSK cells from normal C57BL/6 mice in the presence or absence of autologous Treg cells under B-cell differentiation conditions. Treg cells, regardless of how they were preactivated in vitro, did not affect the differentiation of LSK cells into B220+ CD19+ B cells (Fig. 4). Therefore, Treg cells appear not to directly stimulate the differentiation of hematopoietic progenitors into B cells. In contrast to the lack of effect of Treg cells, the addition of activated CD4+ Foxp3− cells (referred to as Teff cells) reduced the emergence of B220+ CD19+ B cells. When Teff cells were physically separated from the LSK cells in transwells they still exerted their suppressive activity on LSK cells, indicating that their action was contact independent. Indeed, the spent medium from Teff cell cultures suppressed the emergence of B cells to an extent comparable to the effect of Teff cells themselves. Importantly, the suppressive effect of Teff C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Our observations up to this point prompted the hypothesis that soluble factors produced by CD4+ Teff cells are abundant in the milieu of K/Bsf BM and perturb normal B lymphopoiesis. To test this notion, we first observed the functional features of CD4+ T cells residing in K/Bsf BM. BM from normal K/B mice contained large numbers of Foxp3− CD4+ and Foxp3+ CD4+ T cells, as previously reported [12]. We found that the K/Bsf BM harbored more CD4+ T cells than the K/B BM, and most of them retained memory/activated phenotypes (data not shown). Given that T cells expressing an Ag-experienced/memory phenotype can predominantly produce cytokine after short-term polyclonal stimulation in vitro [29], we assessed whether the CD4+ T cells in K/Bsf BM produced hematopoietic cytokines upon TCR/CD28 stimulation. The proportion of cells expressing proinflammatory cytokines such as IFN-γ, TNF, and GM-CSF among the CD4+ T cells was significantly greater in the K/Bsf BM than in the K/B BM (Fig. 5A and B). The proportion of IL-17-expressing cells among the CD4+ T cells was not significantly different in the two strains. The K/Bsf BM contained more IL-6-producing cells, but they were predominantly CD4− rather than CD4+ . The populations of IFN-γ- and TNF-producing cells among the CD4− cells were also larger in the K/Bsf BM. Consistent with these results, supernatants of K/Bsf BM cells stimulated with PMA plus ionomycin contained significantly higher concentrations of IL-6, TNF, and GM-CSF than those of K/B BM cells (Fig. 5C). We next asked whether more of these cytokines are actually produced in vivo in the BM of K/Bsf mice than in the BM of K/B mice. To answer this question, we extracted BM from Brefeldin Atreated mice and counted cytokine-producing cells without in vitro stimulation. We found that the percentages of cells expressing the cytokines (Fig. 5A and B) were substantially higher in K/Bsf BM than in K/B BM, although the difference was less than in the case of in vitro stimulation (Fig. 5D and E). The TNF-producing cells were both CD4+ and CD4− . The IFN-γ-producing cells were predominantly CD4+ rather than CD4− cells, whereas the opposite was true for the IL-6- and GM-CSF-producing cells. These results demonstrate that K/Bsf BM contains higher levels of proinflammatory cytokines derived from Teff cells and/or CD4− cells than K/B BM.
IL-6, TNF, and GM-CSF promote in vitro granulopoiesis at the expense of B lymphopoiesis We asked whether the cytokines whose levels were increased in the K/Bsf BM could suppress B lymphopoiesis. LSK cells from www.eji-journal.eu
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Figure 5. The cytokine milieu of the BM of K/Bsf mice is different from that of K/B mice. (A and B) BM cells from K/B and K/Bsf mice were stimulated with anti-CD3, anti-CD28 mAbs for 6 h, followed by flow cytometry analysis. (C) BM cells from K/B and K/Bsf mice were stimulated with PMA and ionomycin for 24 h, and supernatants were collected to measure levels of cytokines by ELISA. (D and E) K/B and K/Bsf mice were injected with Brefeldin A and assayed postmortem by flow cytometry. (A and D) Representative FACS profiles within a live cell gate and percentages of cytokine-producing cells are shown. (B and E) The percentages of cytokine-producing cells among CD4+ cells and non-CD4+ cells and (C) the levels of cytokines produced by BM cells from K/B () and K/Bsf () mice are shown as mean + SEM ((B) n = 9, (C) n = 4, and (E) n = 5 samples). Data are pooled from (Band C) three, or (E) two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t-test.
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Figure 6. IL-6, TNF, and GM-CSF arrest B lymphopoiesis and promote granulopoiesis. LSK cells purified from C57BL/6 mice were cultured for 12 days under B-cell differentiation conditions in the presence or absence of cytokines as indicated. Differentiated B cells and granulocytes were analyzed by flow cytometry, gated on whole live cells. (A and B) FACS profiles are representative of three independent experiments. (C and D) Data are shown as mean + SEM percentages of the indicated populations and are pooled from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001; Student’s t-test.
normal mice were cultured under B-cell differentiation conditions in the presence of these cytokines. The addition of IL-6, TNF, or GM-CSF almost completely blocked the development of B220+ CD19− and B220+ CD19+ cells, and induced the emergence of B220− Gr-1+ cells even under B-cell differentiation conditions (Fig. 6). IFN-γ reduced the percentage of B220+ CD19+ cells but increased the percentage of B220+ CD19− , pointing to arrest at the pre–pro B-cell stage, and this phenomenon was not accompanied by enhanced development of myeloid cells. IL-17 did not significantly alter the development of either cell line. These results suggest that the elevated concentrations of IL-6, TNF, and/or GMCSF in K/Bsf BM prevent the development of B lymphocytes and conversely enhance granulopoiesis, whereas the inhibitory effect of IFN-γ is restricted to B-lineage cells.
suppressed the development of CD19+ B220+ cells and enhanced the development of B220− Gr-1+ cells, as shown above. The addition of Abs to a single cytokine slightly altered the developmental profiles of the two populations. However, adding Abs to all three cytokines more effectively restored the development of CD19+ B220+ cells and inhibited the development of B220− Gr-1+ cells (Fig. 7A–C). These effects depended on the presence of Teff cells because the same Abs did not interfere with the developmental profiles of LSKs when cultured without Teff cells (Supporting Information Fig. 2). This in vitro finding was confirmed in vivo: administration of these Abs to K/Bsf mice reversed their BM defects, such as reduced CLPs and increased ST-HSCs (Fig. 7D). In the light of these in vitro and in vivo findings, we conclude that the ability of BM Teff cells to inhibit B lymphopoiesis is largely dependent on their secretion of myelopoietic cytokines such as TNF, IL-6, and GM-CSF.
TNF, IL-6, and GM-CSF produced by Teff cells collaborate to inhibit B-cell development
Discussion To see whether the effect of Teff cells on B lymphopenia in the BM is due to IL-6, TNF, and/or GM-CSF, we added neutralizing antibodies to one or more of these cytokines to cocultures of LSK cells and activated Teff cells. Without any additives, Teff cells C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Previously it has been demonstrated that the basal activity of BM is not sufficient for normal hematopoiesis under steady-state conditions, and that hematopoiesis requires the activity of CD4+ T cells www.eji-journal.eu
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Figure 7. Cytokines secreted by activated Teff cells inhibit B lymphopoiesis. (A–C) LSK cells purified from CD45.1+ C57BL/6 mice were cultured alone or together with activated Teff cells from CD45.2+ Foxp3gfp reporter mice under B-cell differentiation conditions in the presence or absence of neutralizing mAb as indicated. After co-culture, differentiated B cells and granulocytes were assessed by FACS, gated on live CD45.1+ cells. (A and B) FACS profiles are representative of three independent experiments. (C) The percentages of B220+ CD19− , B220+ CD19+ , and B220− Gr-1+ CD45.1+ cells in the different conditions are shown. (D) K/Bsf and K/B mice were injected with a mixture of neutralizing mAbs or rat IgG and analyzed by flow cytometry. Cell numbers of each population are displayed as mean + SEM (n = 3 per strain), and data are pooled from two independent experiments. *p < 0.05 and **p < 0.01; Student’s t-test.
constantly activated by BM Ags [30]. Thus, CD4+ T cells in the BM seem to play an indispensable role in normal hematopoiesis, in addition to emergency hematopoiesis. However, it remained unclear whether CD4+ Treg cells are also necessary for normal hematopoiesis, although they are very numerous in the BM. To address this issue, we sought to answer the question whether the absence of Foxp3+ cells impinges on normal C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
hematopoiesis, using a line of viable scurfy mice named K/Bsf. We found that, unlike thymocyte development, which is governed by Foxp3 functions intrinsic to thymic stromal cells [26], B lymphopoiesis in the BM is under the control of Foxp3 functions intrinsic to Treg cells. This finding also implies that Foxp3+ mesenchymal stromal cells residing in BM do not affect hematopoiesis in a Foxp3-dependent manner. We also found that hematopoiesis www.eji-journal.eu
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in Treg-deficient BM was arrested at the ST-HSC stage, and that the progenitors generated committed lineages biased toward myeloid cell fates rather than lymphoid cell fates. In addition, the ratio of GMPs to MEPs was also biased. These results suggest that the activity of Treg cells is essential at several checkpoints in the developmental pathways, including the conversion of the most primitive progenitors to LMPPs. Despite the severe impairment of B lymphopoiesis in the K/Bsf BM, we found that a few pro-B cells still emerged, and these were mostly at the fraction A stage unlike those in the K/B BM. This phenomenon may be due to an additional developmental arrest at the fraction A stage of pre-B cells. However, in the light of a previous report that 20–40% of B220lo CD19− cells are NK cell progenitors [31], we cannot rule out the possibility that the increase in fraction A cells (B220lo IgM− CD43+ HSA− BP-1− ) in K/Bsf BM reflects an increase in NK cell progenitors. An interesting finding is that K/Bsf BM contained more GMPs than K/B BM, despite an approximately 2.4-fold reduction in the number of CMPs—their immediate progenitors. This may indicate that the deficiency of Treg cells leads the CMPs to give rise predominantly to GMPs at the expense of the MEP fate. It is also possible that an alternative pathway to developing GMPs was activated in the K/Bsf BM, and we suspect LMPPs were the progenitors, since they were previously reported to give rise not only to CLPs but also to GMPs [32]. If that is true, it may mean that Foxp3 deficiency licenses LMPPs to preferentially differentiate to GMPs at the expense of the alternative fate: differentiation to CLPs. These data also explain how the rate of reduction of CLP numbers was much greater than that of LMPPs in the K/Bsf mice. However, the observed increase in GMPs and myeloid cells is in conflict with the report of Chen et al. [33], who observed pancytopenia including myeloid cell types, although both studies are most likely in agreement regarding the content of ST-HSCs, MPPs, and CLPs in scurfy BM. This discrepancy may stem from differences in the environmental factors the mice were exposed to, e.g. the type of commensal microbiota, that may interfere with processes genetically imprinted by the Foxp3 mutation. Unlike the regulation of myeloid lineages demonstrated previously [24], the mechanism underlying the effect of Treg cells seems not to include their direct action on progenitors because they did not directly alter the developmental fate of LSK cells cultured under B-cell-polarizing conditions. Rather, Treg cells exerted their effects by constraining the activity of Teff cells. Apart from their action on Treg cells, Teff cells directly affect B lymphopoiesis through the cytokines they produce. Thus, the mechanism by which marrow Treg cells protect normal B lymphopoiesis seems to largely rely on suppressing cytokine production by Teff cells. We identified IL-6, TNF, and GM-CSF as the main cytokines involved in this process, for several reasons. First, their levels were found to be higher in the K/Bsf BM cell cultures than in the K/B BM cells. Second, each could inhibit the differentiation of LSK cells into B cells in vitro. Lastly, a mixture of neutralizing mAbs not only substantially reduced the effect of Teff cells on B lymphopoiesis but also reversed the hematopoietic defects on early B-cell progenitors evident in the K/Bsf BM. Indeed, our results concerning the action C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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of these cytokines are in line with previous studies demonstrating that they can serve as potent hematopoietins elevating myeloid production at the expense of lymphoid production in the setting of chronic inflammation [18–20]. In contrast to previous studies, which mostly focused on a single cytokine, we evaluated each of these cytokines separately but in parallel, at the time of setting up cultures, and found that their inhibitory action on B lymphopoiesis was quite similar, and additive. Intriguingly, the action of IFN-γ, whose level was also enhanced in the K/Bsf BM, was different from the action of the cytokines mentioned above. Like them, IFN-γ interfered with the maturation of B cells, in line with previous observations indicating that it has a negative effect on B lymphopoiesis [34, 35]; however, surprisingly, this was not coupled with enhanced emergence of myeloid-lineage cells. This indicates that promoting myeloid development does not necessarily compromise lymphoid development, and prompts us to classify hematopoietins into two types. One includes IL-6, TNF, and GM-CSF, which not only inhibit B lymphopoiesis but also promote myelopoiesis. The other includes IFN-γ, characterized by uncoupling of the control of these alternative fates. Treg cells appear to inhibit the production of both types of proinflammatory cytokines by Teff cells. Conversely, it has been shown that certain proinflammatory cytokines can dampen the activity of Treg cells by downregulating Foxp3 expression. Indeed, more Treg cells stop expressing Foxp3 (i.e. the so-called exFoxp3 cells) in inflamed tissues under autoimmune conditions than in the steady state [36]. IL-6 and TNF have been shown to convert Treg cells to exFoxp3 cells [37, 38]. These exFoxp3 cells lose their regulatory functions and even produce proinflammatory cytokines such as IFN-γ and IL-17. Moreover, they can be pathogenic when adoptively transferred to normal mice [36]. In this context, both the turnover of Treg cells and the emergence of exFoxp3 cells are expected to be part of a mechanism that mediates the inflammation-driven perturbation of normal hematopoiesis, similar to that seen in scurfy mice. In conclusion, we demonstrate here that Treg cells ensure B lymphopoiesis in the BM by suppressing cytokine production by Teff cells. We suggest a novel role for marrow Treg cells in B-cell homeostasis. In addition, given that lymphopenia-driven homeostatic proliferation is known to cause autoimmune disorders [39], our findings provide a novel mechanism by which Treg cells suppress the development of these diseases.
Materials and methods Mice CD45.1+ and RAG2−/− congenic C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Foxp3gfp reporter mice and KRN transgenic C57BL/6 (K/B) were donated by Drs. Talal A. Chatila and Diane Mathis (Harvard Medical School, MA, USA), respectively [40, 41]. Female C57BL/6 mice carrying a scurfy allele were crossed with male K/B mice to obtain male transgenic scurfy hemizygotes (referred to as K/Bsf). www.eji-journal.eu
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All mice were maintained in a specific pathogen-free facility at Hanyang University. Mice aged 6–7 weeks were used, unless stated otherwise. To measure in vivo cytokine-producing cells, mice were injected intraperitoneally with Brefeldin A (eBioscience) at 250 μg/mouse, and assayed 6 h later postmortem by intracellular flow cytometry methods. In some experiments, mice were injected intravenously with a mixture of mAbs to IL-6, TNF, and GM-CSF (each at 100 μg/mouse/injection; all purchased from BioLegend, San Diego, CA, USA), or rat IgG as a control on days 0 and 2, followed by flow cytometry analysis on day 7. The study was approved by the Institutional Animal Care and Use Committee (Approval ID: HY-IACUC-09-003 and HY-IACUC-12-041).
Flow cytometry and cell sorting Single-cell suspensions of mouse spleen, LNs, and BM cells were surface-stained or intracellularly stained as previously described [42]. To observe cytokine production by BM cells, cells were stimulated for 6 h in the presence of 10 μg/mL anti-CD3 mAb (BD Biosciences, San Diego, CA, USA), 1 μg/mL anti-CD28 mAb (BD Biosciences), 20 U/mL IL-2 (Peprotech, Rocky Hill, NJ, USA) and Golgi-stop (BD Biosciences), and stained with an appropriate mixture of mAbs. To purify Lin− Sca-1+ c-Kit+ (LSK) cells, BM cells were stained with anti-Sca-1, anti-c-Kit, and lineage mixture including mAbs to CD3, B220, Gr-1, Mac-1, and TER-119 (Invitrogen, Carlsbad, CA, USA), followed by flow cytometry. To sort Treg cells and Teff cells, CD4+ T cells were first purified from Foxp3gfp reporter mice by magnetic bead-activated cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany) and were further sorted into GFP+ CD4+ CD25+ populations (Treg cells) and GFP− CD4+ CD25− populations (Teff cells) using a FACS Aria III sorter (BD Biosciences). All isolated cell populations were more than 95% pure. The mAbs used for FACS were described previously [43] and anti-CD34 (RAM34), anti-Flt3 (12B6), anti-TNF (MP6-XT22), anti-IFN-γ (XMG1.2), anti-IL-17A (17B7), anti-IL-6 (MP5–20F3), and anti-GM-CSF (MP1–22E9) were purchased from BD Biosciences or eBiosciences.
T-cell reconstitution into K/Bsf mice CD4+ CD25hi cells and CD4+ CD25− cells from CD45.1+ C57BL/6 mice were purified by MACS. Each cell fraction was more than 95% pure, and nearly all cells in the CD4+ CD25hi fraction were Foxp3+ (>96%). The purified cells were injected intravenously into 5-week-old K/Bsf mice at 3 × 106 cells/mouse. The mice were killed 3 weeks later, and the cells in the BM were examined by flow cytometry.
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cells among the remaining BM cells. CD45.2+ RAG2−/− mice, which had received 500 cGy γ-irradiation 1 day before the transplantation, were injected intravenously with 8 × 106 BM cells from CD45.1+ C57BL/6 mice, 8 × 106 BM cells from CD45.2+ K/Bsf mice, or a mixture of both BM cell populations in a 1:1 ratio. In some experiments, a mixture of 8 × 106 BM cells from CD45.2+ K/Bsf mice and 3 × 106 Treg cells from CD45.1+ C57BL/6 mice was used as donor cells. Recipient mice were sacrificed 6 weeks later, and the BM cells were examined by flow cytometry.
In vitro differentiation of BM cells to B cells OP-9 stromal cells received 1000 cGy γ-irradiation and were seeded in 24-well plates at 6.6 × 103 cells/well in αMEM supplemented with 20% FBS. After 1 day, the medium was changed to RPMI1640 supplemented with 10% FBS, and 1 × 104 /well LSK cells purified from 5-week-old K/B or K/Bsf mice and CD45.1+ C57BL/6 mice were added. To provide B-cell differentiation conditions, cells were cultured in the presence of IL-7, SCF, and Flt3L (all at 20 ng/mL; all purchased from Peprotech) for a total of 12 days. In some experiments, Treg cells or Teff cells that had been preactivated for 3 days with 10 μg/mL anti-CD3 mAb, 1 μg/mL anti-CD28 mAb, and 20 U/mL IL-2 were added to the culture of LSK plus OP-9 cells. Transwell membranes with 0.4 μm pores were used to physically separate the T cells from LSK cells, or alternatively supernatants of cultures of activated Teff cells were added to the LSK culture, making up to 10% of the total culture volume. To assess the effects of cytokines, several cytokines such as IL-6, IL-17, IFN-γ, TNF, and GM-CSF (all at 10 ng/mL; all purchased from Peprotech) were added to the LSK culture. To neutralize cytokines in the LSK culture, anti-IL-6 mAb, antiTNF mAb, anti-GM-CSF mAb, or a mixture of all mAbs (all at 50 μg/mL; all purchased from BioLegend) were added to the LSK culture.
ELISA BM cells were stimulated with 20 ng/mL PMA and 1 μM ionomycin (Sigma-Aldrich, St. Louis, MO, USA), and supernatants were collected after 24 h of culture. Cytokine concentrations in the culture supernatants were measured by sandwich ELISAs using BD OptEIATM kits (BD Biosciences) according to the manufacturer’s instructions.
Statistical analyses Data are presented as mean ± SEM. Differences between groups were evaluated by unpaired Student’s t-tests. p Values are reported for statistically significant differences between groups (
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Foxp3+ regulatory T cells ensure B lymphopoiesis by inhibiting the granulopoietic activity of effector T cells in mouse bone marrow Sunghoon Kim ∗1 , Kyungsoo Park ∗2 , Jinwook Choi2 , Eunkyeong Jang3 , Doo-Jin Paik3 , Rho H. Seong ∗∗2 and Jeehee Youn ∗∗1,3 1
Department of Biomedical Sciences, Hanyang University Graduate School, Seoul, Korea Department of Biological Sciences, Research Center for Functional Cellulomics, Seoul National University, Seoul, Korea 3 Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul, Korea 2
Foxp3+ Treg cells are crucial for maintaining T-cell homeostasis, but their role in B-cell homeostasis remains unclear. Here, we found that Foxp3 mutant scurfy mice had fewer B-lineage cells and progenitors, including common lymphoid progenitors and lymphoidprimed multipotent progenitors, but higher myeloid-lineage cell numbers in BM compared with WT littermates. Homeostasis within the HSC compartment was also compromised with apparent expansion of long- and short-term HSCs. This abnormality was due to the lack of Treg cells, but not to the Treg-cell extrinsic functions of Foxp3 or cellautonomous defects. Among cytokines enriched in the BM of scurfy mice, IFN-γ affected only B lymphopoiesis, but GM-CSF, TNF, and IL-6 collectively promoted granulopoiesis at the expense of B lymphopoiesis. Neutralization of these three cytokines reversed the hematopoietic defects on early B-cell progenitors in scurfy mice. Treg cells ensured B lymphopoiesis by reducing the production of these cytokines by effector T cells, but not by directly affecting B lymphopoiesis. These results suggest that Treg cells occupy an important niche in the BM to protect B-lineage progenitor cells from excessive exposure to a lymphopoiesis-regulating milieu.
Keywords: B lymphopoiesis r Foxp3
r
Granulopoietins r Hematopoiesis r Regulatory T cells
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction CD4+ Foxp3+ Treg cells are essential for maintaining immune homeostasis, as evidenced by the severe autoimmune lymphoproliferative disease observed in Treg-cell-deficient mice and humans [1, 2]. Treg cells were initially identified in the secondary lymphoid tissues where they suppress the initial priming and
Correspondence: Prof. Jeehee Youn e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
effector differentiation of autoreactive T cells [3]. They function by directly suppressing T cells and/or interacting with DCs to limit their ability to prime T cells effectively [4, 5]. In addition to their constitutive presence in secondary lymphoid tissues, Treg cells have been found in several nonlymphoid sites, such as mucosal tissue, skin, liver, and lung, during inflammation as well as in steady-state conditions [6–10]. These Treg cells appear to regulate
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These authors contributed equally to this work. These senior authors contributed equally to this work.
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tissue-tropic autoimmunity and inflammation, and alter pathogen clearance during peripheral infection. A diverse array of adhesion molecules and chemokine receptors that are expressed on Treg cells seem to license them to migrate to peripheral tissues. A fraction of Treg cells have been shown to express the BM homing receptor CXCR4 [11], suggesting the existence of a niche for Treg cells in the BM. Indeed, there are studies showing that healthy human BM contains a substantial number of Treg cells that traffic via CXCL12/CXCR4 signaling [12]. However, little is known of the role and significance of BM-residing Treg cells in the context of BM homeostasis. BM harbors a variety of hematopoietic lineage cells, including lymphoid and myeloid lineage cells [13–15]. All hematopoietic cells are derived from pluripotent HSCs. Long-term (LT)-HSCs lack all lineage markers (Lin− ) but express high levels of Sca-1 and c-Kit on their surface and give rise sequentially to short-term (ST)-HSCs and multipotent progenitors (MPPs), both of which are characterized by a loss of self-renewal capacity. MPPs are the most primitive progenitors, bifurcating into distinct lineage fates, namely common myeloid progenitors (CMPs) and lymphoidprimed multipotent progenitors (LMPPs). While CMPs give rise to granulocyte-monocyte progenitors (GMPs) and megakaryocyteerythrocyte progenitors (MEPs), LMPPs preferentially differentiate into common lymphoid progenitors (CLPs), a population of early lymphoid progenitors that has a reduced level of c-Kit and displays surface IL-7 receptor α chain. CLPs undergo a cascade of highly ordered steps until immature B cells emerge in the BM. After exit from the BM, immature B cells continue their maturation in the secondary lymphoid tissues to form the peripheral immune repertoire. Hematopoiesis is normally well controlled in order to replenish blood cells at a constant rate, such that the balance of lymphoid and myeloid cells is maintained. However, the output of particular blood cell types can be altered under certain conditions. For example, acute infections or acute allergic responses elevate the production of granulocytes at the expense of B lymphopoiesis, a phenomenon known as “emergency granulopoiesis” [16, 17]. This seems to be at least in part mediated by proinflammatory cytokines such as GM-CSF, TNF, and IL-6, since they are known to shift the developmental pathway of BM progenitors toward granulopoiesis [15, 18–20]. Whether the activity of Foxp3+ Treg cells also influences hematopoiesis in the BM remains a matter of debate, since contradictory roles of Treg cells in hematopoiesis have been reported, specifically in B-cell development in the BM [21–23]. Although it was reported that Treg cells can directly inhibit in vitro differentiation of myeloid progenitor cells in a contact-dependent manner, and this effect was corroborated in vivo when Treg cells were cotransferred with BM cells into immunocompromised mice [24], it was not clear whether this mechanism also applies to lymphoid progenitors. Moreover, the demonstration that administration of Treg cells could not rescue the defective B-cell development in scurfy mice [25] implies that the B lymphopenia in scurfy BM is independent of the deficiency in Treg cells. Therefore, although most, but not all, previous studies detected a defect in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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B lymphopoiesis in scurfy BM, the underlying mechanism remained to be elucidated. Here, we analyzed all the aspects of BM hematopoiesis, including B lymphopoiesis, in a viable line of scurfy mice and revealed the mechanism by which Treg cells regulate the composition of BM cell populations. Our in vivo and in vitro data, taken together, suggest that effector T (Teff) cells infiltrating the BM perturb normal B lymphopoiesis by producing diverse hematopoiesis-regulating cytokines, and that Treg cells present in the BM protect B-lineage progenitor cells from this activity of Teff cells but do not directly regulate the activity of hematopoietic progenitor cells.
Results Perturbed hematopoiesis in the BM of Foxp3-deficient mice Scurfy mice harboring a loss-of-function mutation of Foxp3 do not develop Foxp3+ Treg cells, and so they can be used to investigate the role of Treg cells in vivo. However, scurfy mice on the C57BL/6 background (referred as to B6sf) usually succumb to extremely severe inflammatory symptoms at about 4 weeks of age, so there are practical difficulties associated with using them. To overcome these, we generated male KRN TCR transgenic C57BL/6 mice bearing a scurfy allele on the X chromosome (referred to as K/Bsf). As expected, K/Bsf mice exhibited much more modest symptoms of inflammation, and about 80% survived until at least 7 weeks of age by which time all our experiments were completed (data not shown). We first determined whether the absence of Foxp3 altered BM hematopoiesis in the K/Bsf mice. BM from K/Bsf mice contained significantly more Gr-1+ Mac-1+ myeloid cells, fewer B220+ B-lineage cells, and fewer TER-119+ erythroid cells than BM from their transgenic nonscurfy littermates (referred to as K/B hereafter; Fig. 1A). We further traced the B-lineage population through all cell stages and found that both percentages and cell numbers of pro-B (B220lo IgM− CD43+ ), pre-B (B220lo IgM− CD43− ), immature B (B220lo IgM+ ) and mature/recirculating B (B220hi IgM+ ) cell populations were lower in K/Bsf BM than in K/B BM (Fig. 1B). Moreover, the ratio of pro-B cells to pre-B cells was higher in K/Bsf BM. Consistent with this observation, the difference of cell numbers between the two strains was largest at the pre-B-cell stage. When we subdivided the B220lo IgM− CD43+ cells into fractions A, B, and C, the frequency and number of cells in fraction A were greater than those in fractions B and C in K/Bsf BM, whereas in K/B BM the cell numbers in all three fractions were similar (Fig. 1C). Next, we examined whether the B lymphopenia observed in K/Bsf BM stemmed from impaired development of earlier progenitors of the B-lineage cells. The number of CLPs (Lin− c-Kitlo Sca-1lo IL-7R+ ) was found to decrease dramatically in K/Bsf BM (Fig. 1D). LMPPs (Lin− Sca-1+ c-Kit+ Flt3+ CD34+ ) were also lower, albeit not as low as CLPs, while LT-HSCs (Lin− Sca-1+ c-Kit+ Flt3− CD34− ) and ST-HSCs (Lin− Sca-1+ c-Kit+ Flt3− CD34+ ) www.eji-journal.eu
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Figure 1. Normal hematopoiesis is perturbed in the BM of K/Bsf mice. BM cells from K/B () and K/Bsf () mice were stained with Abs to lineagespecific markers and analyzed by flow cytometry. (A) Myeloid lineages (Mac-1+ Gr-1+ ), erythroid cells (TER 119+ ), and B-lineage cells (B220+ ) gated on whole live cells. (B) Pro-B plus pre-B cells (B220lo IgM− ), immature B cells (B220lo IgM+ ), and mature B cells (B220hi IgM+ ) within a live cell gate (top plots). Pro-B cells (B220+ CD43+ ) and pre-B cells (B220+ CD43− ) within a B220lo IgM− gate (middle plots). (C) Pre–pro B cells (fraction A, HSA− BP-1− ), early pro-B cells (fraction B, HSA+ BP-1− ) and late pro-B cells (fraction C, HSA+ BP-1+ ) gated on B220+ CD43+ IgM− cells. (D) CLP population (Lin− Sca-1low c-Kitlow ) gated on Lin− IL-7Rα+ cells. (E) LT-HSC (Lin− Sca-1+ c-Kit+ Flt3− CD34− ), ST-HSC (Lin− Sca-1+ c-Kit+ Flt3− CD34+ ), and LMPP (Lin− Sca1+ c-Kit+ Flt3+ CD34+ ) gated on Lin− Sca-1+ c-Kit+ cells. (F) CMP (Lin− Sca-1− c-Kit+ CD34+ CD16/32− ), GMP (Lin− Sca-1− c-Kit+ CD34+ CD16/32+ ), and MEP (Lin− Sca-1− c-Kit+ CD34− CD16/32− ) populations gated on Lin− Sca-1− c-Kit+ cells. (A–F) Representative FACS profiles and mean + SEM cell numbers (n = 6 mice/group) pooled from four independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t-test.
were increased (Fig. 1E). These results indicate that the impairment of B-cell development in Foxp3-deficient mice starts with developmental arrest at the most primitive progenitors, namely LT-HSCs and ST-HSCs. In another set of staining experiments to detect the alternative fate of ST-HSC differentiation, CMPs were found to be about 2.4-fold less frequent in K/Bsf BM than in K/B BM, indicating that the Foxp3 deficiency-dependent arrest at the stage of ST-HSCs also blocks the emergence of CMPs, as well as LMPPs (Fig. 1F). Interestingly, despite this reduction, there were more GMPs in K/Bsf BM than in K/B BM, while the opposite was true for MEPs. We confirmed that there were no significant differences between K/Bsf and B6sf mice in terms of the cellular profiles of primary and secondary immune compartments. Moreover, all the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
aspects of K/Bsf BM mentioned above were precisely mirrored in B6sf BM, indicating that expression of the KRN transgene did not affect hematopoiesis in the scurfy mice (Supporting Information Fig. 1).
K/Bsf BM cells impaired hematopoiesis is due to Treg-cell deficiency, but is independent of Foxp3 We examined the mechanism by which hematopoiesis, in particular B lymphopoiesis, is impaired in the K/Bsf mice. Because previous studies had demonstrated Foxp3 expression in non-Treg cells, such as BM-derived mesenchymal stromal cells, thymic stromal cells, and mammary cells [26–28], we first needed to address www.eji-journal.eu
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Figure 2. Developmental profiles of K/Bsf BM cells in vitro and in BM chimeric mice. (A) LSK cells purified from K/B and K/Bsf mice were cultured on OP-9 stromal cells in the presence of IL-7, SCF, and Flt3L for 12 days and analyzed by flow cytometry, gated on whole live cells. Representative FACS profiles and percentages of each cell population derived from K/B and K/Bsf LSK cells are shown. (B–F) T-cell-depleted BM cells from CD45.1+ C57BL/6 mice, T-cell-depleted BM cells from CD45.2+ K/Bsf mice, or a mixture of both BM cells were transferred into sublethally irradiated RAG2−/− recipients. Six weeks after transplantation, B-lineage populations in the recipient BM were examined by flow cytometry. Percentages of B-lineage cells among (B and C) donor-derived whole BM cells, (D) donor-derived B220lo IgM− BM cells, and (E) donor-derived B220lo IgM− CD43+ BM cells are shown. (F) Total cell numbers in the recipient BM are shown. (A–F) Data are presented as mean + SEM (n = 3 mice/group) and are representative of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001; Student’s t-test.
the question of whether the impairment could be attributed to the Treg-cell-extrinsic function of Foxp3. To this end, LSK cells (Lin− Sca-1+ c-Kit+ ) purified from K/Bsf mice and K/B mice were cultured under B-cell differentiation conditions for 12 days, and the phenotypes of the resulting cells were analyzed by flow cytometry. LSK cells from K/Bsf mice formed B220+ CD19− pre–pro B cells and the more mature B220+ CD19+ B cells to an extent similar to that observed in the K/B mice (Fig. 2A). Thus, LSK cells from the K/Bsf mice seemed to be unaffected in their potential to give rise to B cells, and the abnormal B lymphopoiesis might, therefore, result from a Foxp3 function extrinsic to the hematopoietic progenitor cells. However, the normal B lymphopoiesis observed in the K/Bsf BM cells in vitro was not observed in mice that received these same C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cells by adoptive transfer: K/Bsf BM cells transplanted into sublethally irradiated RAG2−/− mice failed to give rise to B-cell subsets, unlike Foxp3-intact congenic CD45.1+ BM cells (Fig. 2B–F). To see whether this defect in B-cell development could be corrected by intact BM cells, we generated mixed BM chimeras by transplanting a mixture of congenic CD45.1+ Foxp3+/+ BM cells (50%) with CD45.2+ K/Bsf BM cells (50%) into irradiated RAG2−/− recipients. In the resulting chimeras, the CD45.2+ K/Bsf BM cells gave rise to all the hematopoietic cell populations including B-lineage cells. This shows that the abnormal B lymphopoiesis in K/Bsf mice is not due to any intrinsic defect in the hematopoietic progenitors, since they give rise to B cells in the presence of some “element” derived from the control BM progenitors.
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Figure 3. Treg cells repair abnormal B lymphopoiesis of BM cells from K/Bsf mice. (A) RAG2−/− mice were infused with T-cell-depleted K/Bsf BM cells alone or together with Treg cells purified from CD45.1+ C57BL/6 mice and assayed 6 weeks later by flow cytometry. Data are shown as mean + SEM of four samples pooled from two independent experiments. (B) CD4+ CD25hi or CD4+ CD25− cells purified from CD45.1+ C57BL/6 mice were injected into K/Bsf mice. Three weeks later, the BM cells of recipient mice were examined by flow cytometry. Data are shown as mean + SEM of five samples and are pooled from three independent experiments. *p < 0.05, **p < 0.01; Student’s t-test.
To determine whether this element was in fact the population of Treg cells, we generated mixed BM chimeras by transferring a mixture of CD45.2+ K/Bsf BM cells with CD45.1+ Treg cells into irradiated RAG2−/− recipients, and found that the defects in B-lineage cell formation of the K/Bsf donors were substantially relieved (Fig. 3A). Thus, Treg cells seemed to be sufficient to ensure that K/Bsf donor cells formed B-lineage cells normally.
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We confirmed this finding by reconstituting K/Bsf mice with Treg cells. The mice were infused with either CD4+ CD25hi cells (presumed to be a Treg-cell population) or CD4+ CD25− cells purified from CD45.1+ C57BL/6 mice. Infusion with CD4+ CD25hi cells greatly reduced all the aberrant phenotypes of K/Bsf BM (Fig. 3B). Taken together, our data indicate that the abnormal hematopoiesis observed in K/Bsf BM stems from the absence of Treg cells rather than from a Treg-cell-independent function of Foxp3.
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cells on B lymphopoiesis was fully inhibited by the addition of Treg cells. Taken together, these results demonstrate that Treg cells protect B lymphopoiesis from the suppressive activity of soluble factors derived from Teff cells.
The cytokine milieu of K/Bsf BM is different from that of normal BM
Figure 4. Treg cells ensure B lymphopoiesis by controlling the suppressive activity of Teff cells. LSK cells purified from CD45.1+ C57BL/6 mice were cultured in the presence or absence of preactivated Treg cells or/and Teff cells purified from Foxp3gfp reporter mice. In some experiments, Teff cells were replaced by the supernatants from cultures of activated Teff cells (Teff sup) or Teff cells were added into transwells (tw). After 12 days of culture, differentiated B cells (B220+ CD19+ ) were examined by flow cytometry, gated on whole live cells. (A) FACS profiles are representative of three independent experiments. (B) Data are shown as mean + SEM percentages of B220+ CD19+ cells and are pooled from three independent experiments. ***p < 0.001; Student’s t-test. NS: not significant.
Treg cells protect B lymphopoiesis by controlling the suppressive activity of Teff cells Our finding that the scurfy-associated perturbation of B lymphopoiesis was due to Treg-cell-autonomous defects in Foxp3 function suggests that Treg cells play a crucial role in B lymphopoiesis in the BM. Whether Treg cells directly control B lymphopoiesis was assessed by culturing LSK cells from normal C57BL/6 mice in the presence or absence of autologous Treg cells under B-cell differentiation conditions. Treg cells, regardless of how they were preactivated in vitro, did not affect the differentiation of LSK cells into B220+ CD19+ B cells (Fig. 4). Therefore, Treg cells appear not to directly stimulate the differentiation of hematopoietic progenitors into B cells. In contrast to the lack of effect of Treg cells, the addition of activated CD4+ Foxp3− cells (referred to as Teff cells) reduced the emergence of B220+ CD19+ B cells. When Teff cells were physically separated from the LSK cells in transwells they still exerted their suppressive activity on LSK cells, indicating that their action was contact independent. Indeed, the spent medium from Teff cell cultures suppressed the emergence of B cells to an extent comparable to the effect of Teff cells themselves. Importantly, the suppressive effect of Teff C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Our observations up to this point prompted the hypothesis that soluble factors produced by CD4+ Teff cells are abundant in the milieu of K/Bsf BM and perturb normal B lymphopoiesis. To test this notion, we first observed the functional features of CD4+ T cells residing in K/Bsf BM. BM from normal K/B mice contained large numbers of Foxp3− CD4+ and Foxp3+ CD4+ T cells, as previously reported [12]. We found that the K/Bsf BM harbored more CD4+ T cells than the K/B BM, and most of them retained memory/activated phenotypes (data not shown). Given that T cells expressing an Ag-experienced/memory phenotype can predominantly produce cytokine after short-term polyclonal stimulation in vitro [29], we assessed whether the CD4+ T cells in K/Bsf BM produced hematopoietic cytokines upon TCR/CD28 stimulation. The proportion of cells expressing proinflammatory cytokines such as IFN-γ, TNF, and GM-CSF among the CD4+ T cells was significantly greater in the K/Bsf BM than in the K/B BM (Fig. 5A and B). The proportion of IL-17-expressing cells among the CD4+ T cells was not significantly different in the two strains. The K/Bsf BM contained more IL-6-producing cells, but they were predominantly CD4− rather than CD4+ . The populations of IFN-γ- and TNF-producing cells among the CD4− cells were also larger in the K/Bsf BM. Consistent with these results, supernatants of K/Bsf BM cells stimulated with PMA plus ionomycin contained significantly higher concentrations of IL-6, TNF, and GM-CSF than those of K/B BM cells (Fig. 5C). We next asked whether more of these cytokines are actually produced in vivo in the BM of K/Bsf mice than in the BM of K/B mice. To answer this question, we extracted BM from Brefeldin Atreated mice and counted cytokine-producing cells without in vitro stimulation. We found that the percentages of cells expressing the cytokines (Fig. 5A and B) were substantially higher in K/Bsf BM than in K/B BM, although the difference was less than in the case of in vitro stimulation (Fig. 5D and E). The TNF-producing cells were both CD4+ and CD4− . The IFN-γ-producing cells were predominantly CD4+ rather than CD4− cells, whereas the opposite was true for the IL-6- and GM-CSF-producing cells. These results demonstrate that K/Bsf BM contains higher levels of proinflammatory cytokines derived from Teff cells and/or CD4− cells than K/B BM.
IL-6, TNF, and GM-CSF promote in vitro granulopoiesis at the expense of B lymphopoiesis We asked whether the cytokines whose levels were increased in the K/Bsf BM could suppress B lymphopoiesis. LSK cells from www.eji-journal.eu
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Figure 5. The cytokine milieu of the BM of K/Bsf mice is different from that of K/B mice. (A and B) BM cells from K/B and K/Bsf mice were stimulated with anti-CD3, anti-CD28 mAbs for 6 h, followed by flow cytometry analysis. (C) BM cells from K/B and K/Bsf mice were stimulated with PMA and ionomycin for 24 h, and supernatants were collected to measure levels of cytokines by ELISA. (D and E) K/B and K/Bsf mice were injected with Brefeldin A and assayed postmortem by flow cytometry. (A and D) Representative FACS profiles within a live cell gate and percentages of cytokine-producing cells are shown. (B and E) The percentages of cytokine-producing cells among CD4+ cells and non-CD4+ cells and (C) the levels of cytokines produced by BM cells from K/B () and K/Bsf () mice are shown as mean + SEM ((B) n = 9, (C) n = 4, and (E) n = 5 samples). Data are pooled from (Band C) three, or (E) two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t-test.
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Figure 6. IL-6, TNF, and GM-CSF arrest B lymphopoiesis and promote granulopoiesis. LSK cells purified from C57BL/6 mice were cultured for 12 days under B-cell differentiation conditions in the presence or absence of cytokines as indicated. Differentiated B cells and granulocytes were analyzed by flow cytometry, gated on whole live cells. (A and B) FACS profiles are representative of three independent experiments. (C and D) Data are shown as mean + SEM percentages of the indicated populations and are pooled from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001; Student’s t-test.
normal mice were cultured under B-cell differentiation conditions in the presence of these cytokines. The addition of IL-6, TNF, or GM-CSF almost completely blocked the development of B220+ CD19− and B220+ CD19+ cells, and induced the emergence of B220− Gr-1+ cells even under B-cell differentiation conditions (Fig. 6). IFN-γ reduced the percentage of B220+ CD19+ cells but increased the percentage of B220+ CD19− , pointing to arrest at the pre–pro B-cell stage, and this phenomenon was not accompanied by enhanced development of myeloid cells. IL-17 did not significantly alter the development of either cell line. These results suggest that the elevated concentrations of IL-6, TNF, and/or GMCSF in K/Bsf BM prevent the development of B lymphocytes and conversely enhance granulopoiesis, whereas the inhibitory effect of IFN-γ is restricted to B-lineage cells.
suppressed the development of CD19+ B220+ cells and enhanced the development of B220− Gr-1+ cells, as shown above. The addition of Abs to a single cytokine slightly altered the developmental profiles of the two populations. However, adding Abs to all three cytokines more effectively restored the development of CD19+ B220+ cells and inhibited the development of B220− Gr-1+ cells (Fig. 7A–C). These effects depended on the presence of Teff cells because the same Abs did not interfere with the developmental profiles of LSKs when cultured without Teff cells (Supporting Information Fig. 2). This in vitro finding was confirmed in vivo: administration of these Abs to K/Bsf mice reversed their BM defects, such as reduced CLPs and increased ST-HSCs (Fig. 7D). In the light of these in vitro and in vivo findings, we conclude that the ability of BM Teff cells to inhibit B lymphopoiesis is largely dependent on their secretion of myelopoietic cytokines such as TNF, IL-6, and GM-CSF.
TNF, IL-6, and GM-CSF produced by Teff cells collaborate to inhibit B-cell development
Discussion To see whether the effect of Teff cells on B lymphopenia in the BM is due to IL-6, TNF, and/or GM-CSF, we added neutralizing antibodies to one or more of these cytokines to cocultures of LSK cells and activated Teff cells. Without any additives, Teff cells C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Previously it has been demonstrated that the basal activity of BM is not sufficient for normal hematopoiesis under steady-state conditions, and that hematopoiesis requires the activity of CD4+ T cells www.eji-journal.eu
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Figure 7. Cytokines secreted by activated Teff cells inhibit B lymphopoiesis. (A–C) LSK cells purified from CD45.1+ C57BL/6 mice were cultured alone or together with activated Teff cells from CD45.2+ Foxp3gfp reporter mice under B-cell differentiation conditions in the presence or absence of neutralizing mAb as indicated. After co-culture, differentiated B cells and granulocytes were assessed by FACS, gated on live CD45.1+ cells. (A and B) FACS profiles are representative of three independent experiments. (C) The percentages of B220+ CD19− , B220+ CD19+ , and B220− Gr-1+ CD45.1+ cells in the different conditions are shown. (D) K/Bsf and K/B mice were injected with a mixture of neutralizing mAbs or rat IgG and analyzed by flow cytometry. Cell numbers of each population are displayed as mean + SEM (n = 3 per strain), and data are pooled from two independent experiments. *p < 0.05 and **p < 0.01; Student’s t-test.
constantly activated by BM Ags [30]. Thus, CD4+ T cells in the BM seem to play an indispensable role in normal hematopoiesis, in addition to emergency hematopoiesis. However, it remained unclear whether CD4+ Treg cells are also necessary for normal hematopoiesis, although they are very numerous in the BM. To address this issue, we sought to answer the question whether the absence of Foxp3+ cells impinges on normal C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
hematopoiesis, using a line of viable scurfy mice named K/Bsf. We found that, unlike thymocyte development, which is governed by Foxp3 functions intrinsic to thymic stromal cells [26], B lymphopoiesis in the BM is under the control of Foxp3 functions intrinsic to Treg cells. This finding also implies that Foxp3+ mesenchymal stromal cells residing in BM do not affect hematopoiesis in a Foxp3-dependent manner. We also found that hematopoiesis www.eji-journal.eu
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in Treg-deficient BM was arrested at the ST-HSC stage, and that the progenitors generated committed lineages biased toward myeloid cell fates rather than lymphoid cell fates. In addition, the ratio of GMPs to MEPs was also biased. These results suggest that the activity of Treg cells is essential at several checkpoints in the developmental pathways, including the conversion of the most primitive progenitors to LMPPs. Despite the severe impairment of B lymphopoiesis in the K/Bsf BM, we found that a few pro-B cells still emerged, and these were mostly at the fraction A stage unlike those in the K/B BM. This phenomenon may be due to an additional developmental arrest at the fraction A stage of pre-B cells. However, in the light of a previous report that 20–40% of B220lo CD19− cells are NK cell progenitors [31], we cannot rule out the possibility that the increase in fraction A cells (B220lo IgM− CD43+ HSA− BP-1− ) in K/Bsf BM reflects an increase in NK cell progenitors. An interesting finding is that K/Bsf BM contained more GMPs than K/B BM, despite an approximately 2.4-fold reduction in the number of CMPs—their immediate progenitors. This may indicate that the deficiency of Treg cells leads the CMPs to give rise predominantly to GMPs at the expense of the MEP fate. It is also possible that an alternative pathway to developing GMPs was activated in the K/Bsf BM, and we suspect LMPPs were the progenitors, since they were previously reported to give rise not only to CLPs but also to GMPs [32]. If that is true, it may mean that Foxp3 deficiency licenses LMPPs to preferentially differentiate to GMPs at the expense of the alternative fate: differentiation to CLPs. These data also explain how the rate of reduction of CLP numbers was much greater than that of LMPPs in the K/Bsf mice. However, the observed increase in GMPs and myeloid cells is in conflict with the report of Chen et al. [33], who observed pancytopenia including myeloid cell types, although both studies are most likely in agreement regarding the content of ST-HSCs, MPPs, and CLPs in scurfy BM. This discrepancy may stem from differences in the environmental factors the mice were exposed to, e.g. the type of commensal microbiota, that may interfere with processes genetically imprinted by the Foxp3 mutation. Unlike the regulation of myeloid lineages demonstrated previously [24], the mechanism underlying the effect of Treg cells seems not to include their direct action on progenitors because they did not directly alter the developmental fate of LSK cells cultured under B-cell-polarizing conditions. Rather, Treg cells exerted their effects by constraining the activity of Teff cells. Apart from their action on Treg cells, Teff cells directly affect B lymphopoiesis through the cytokines they produce. Thus, the mechanism by which marrow Treg cells protect normal B lymphopoiesis seems to largely rely on suppressing cytokine production by Teff cells. We identified IL-6, TNF, and GM-CSF as the main cytokines involved in this process, for several reasons. First, their levels were found to be higher in the K/Bsf BM cell cultures than in the K/B BM cells. Second, each could inhibit the differentiation of LSK cells into B cells in vitro. Lastly, a mixture of neutralizing mAbs not only substantially reduced the effect of Teff cells on B lymphopoiesis but also reversed the hematopoietic defects on early B-cell progenitors evident in the K/Bsf BM. Indeed, our results concerning the action C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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of these cytokines are in line with previous studies demonstrating that they can serve as potent hematopoietins elevating myeloid production at the expense of lymphoid production in the setting of chronic inflammation [18–20]. In contrast to previous studies, which mostly focused on a single cytokine, we evaluated each of these cytokines separately but in parallel, at the time of setting up cultures, and found that their inhibitory action on B lymphopoiesis was quite similar, and additive. Intriguingly, the action of IFN-γ, whose level was also enhanced in the K/Bsf BM, was different from the action of the cytokines mentioned above. Like them, IFN-γ interfered with the maturation of B cells, in line with previous observations indicating that it has a negative effect on B lymphopoiesis [34, 35]; however, surprisingly, this was not coupled with enhanced emergence of myeloid-lineage cells. This indicates that promoting myeloid development does not necessarily compromise lymphoid development, and prompts us to classify hematopoietins into two types. One includes IL-6, TNF, and GM-CSF, which not only inhibit B lymphopoiesis but also promote myelopoiesis. The other includes IFN-γ, characterized by uncoupling of the control of these alternative fates. Treg cells appear to inhibit the production of both types of proinflammatory cytokines by Teff cells. Conversely, it has been shown that certain proinflammatory cytokines can dampen the activity of Treg cells by downregulating Foxp3 expression. Indeed, more Treg cells stop expressing Foxp3 (i.e. the so-called exFoxp3 cells) in inflamed tissues under autoimmune conditions than in the steady state [36]. IL-6 and TNF have been shown to convert Treg cells to exFoxp3 cells [37, 38]. These exFoxp3 cells lose their regulatory functions and even produce proinflammatory cytokines such as IFN-γ and IL-17. Moreover, they can be pathogenic when adoptively transferred to normal mice [36]. In this context, both the turnover of Treg cells and the emergence of exFoxp3 cells are expected to be part of a mechanism that mediates the inflammation-driven perturbation of normal hematopoiesis, similar to that seen in scurfy mice. In conclusion, we demonstrate here that Treg cells ensure B lymphopoiesis in the BM by suppressing cytokine production by Teff cells. We suggest a novel role for marrow Treg cells in B-cell homeostasis. In addition, given that lymphopenia-driven homeostatic proliferation is known to cause autoimmune disorders [39], our findings provide a novel mechanism by which Treg cells suppress the development of these diseases.
Materials and methods Mice CD45.1+ and RAG2−/− congenic C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Foxp3gfp reporter mice and KRN transgenic C57BL/6 (K/B) were donated by Drs. Talal A. Chatila and Diane Mathis (Harvard Medical School, MA, USA), respectively [40, 41]. Female C57BL/6 mice carrying a scurfy allele were crossed with male K/B mice to obtain male transgenic scurfy hemizygotes (referred to as K/Bsf). www.eji-journal.eu
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All mice were maintained in a specific pathogen-free facility at Hanyang University. Mice aged 6–7 weeks were used, unless stated otherwise. To measure in vivo cytokine-producing cells, mice were injected intraperitoneally with Brefeldin A (eBioscience) at 250 μg/mouse, and assayed 6 h later postmortem by intracellular flow cytometry methods. In some experiments, mice were injected intravenously with a mixture of mAbs to IL-6, TNF, and GM-CSF (each at 100 μg/mouse/injection; all purchased from BioLegend, San Diego, CA, USA), or rat IgG as a control on days 0 and 2, followed by flow cytometry analysis on day 7. The study was approved by the Institutional Animal Care and Use Committee (Approval ID: HY-IACUC-09-003 and HY-IACUC-12-041).
Flow cytometry and cell sorting Single-cell suspensions of mouse spleen, LNs, and BM cells were surface-stained or intracellularly stained as previously described [42]. To observe cytokine production by BM cells, cells were stimulated for 6 h in the presence of 10 μg/mL anti-CD3 mAb (BD Biosciences, San Diego, CA, USA), 1 μg/mL anti-CD28 mAb (BD Biosciences), 20 U/mL IL-2 (Peprotech, Rocky Hill, NJ, USA) and Golgi-stop (BD Biosciences), and stained with an appropriate mixture of mAbs. To purify Lin− Sca-1+ c-Kit+ (LSK) cells, BM cells were stained with anti-Sca-1, anti-c-Kit, and lineage mixture including mAbs to CD3, B220, Gr-1, Mac-1, and TER-119 (Invitrogen, Carlsbad, CA, USA), followed by flow cytometry. To sort Treg cells and Teff cells, CD4+ T cells were first purified from Foxp3gfp reporter mice by magnetic bead-activated cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany) and were further sorted into GFP+ CD4+ CD25+ populations (Treg cells) and GFP− CD4+ CD25− populations (Teff cells) using a FACS Aria III sorter (BD Biosciences). All isolated cell populations were more than 95% pure. The mAbs used for FACS were described previously [43] and anti-CD34 (RAM34), anti-Flt3 (12B6), anti-TNF (MP6-XT22), anti-IFN-γ (XMG1.2), anti-IL-17A (17B7), anti-IL-6 (MP5–20F3), and anti-GM-CSF (MP1–22E9) were purchased from BD Biosciences or eBiosciences.
T-cell reconstitution into K/Bsf mice CD4+ CD25hi cells and CD4+ CD25− cells from CD45.1+ C57BL/6 mice were purified by MACS. Each cell fraction was more than 95% pure, and nearly all cells in the CD4+ CD25hi fraction were Foxp3+ (>96%). The purified cells were injected intravenously into 5-week-old K/Bsf mice at 3 × 106 cells/mouse. The mice were killed 3 weeks later, and the cells in the BM were examined by flow cytometry.
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cells among the remaining BM cells. CD45.2+ RAG2−/− mice, which had received 500 cGy γ-irradiation 1 day before the transplantation, were injected intravenously with 8 × 106 BM cells from CD45.1+ C57BL/6 mice, 8 × 106 BM cells from CD45.2+ K/Bsf mice, or a mixture of both BM cell populations in a 1:1 ratio. In some experiments, a mixture of 8 × 106 BM cells from CD45.2+ K/Bsf mice and 3 × 106 Treg cells from CD45.1+ C57BL/6 mice was used as donor cells. Recipient mice were sacrificed 6 weeks later, and the BM cells were examined by flow cytometry.
In vitro differentiation of BM cells to B cells OP-9 stromal cells received 1000 cGy γ-irradiation and were seeded in 24-well plates at 6.6 × 103 cells/well in αMEM supplemented with 20% FBS. After 1 day, the medium was changed to RPMI1640 supplemented with 10% FBS, and 1 × 104 /well LSK cells purified from 5-week-old K/B or K/Bsf mice and CD45.1+ C57BL/6 mice were added. To provide B-cell differentiation conditions, cells were cultured in the presence of IL-7, SCF, and Flt3L (all at 20 ng/mL; all purchased from Peprotech) for a total of 12 days. In some experiments, Treg cells or Teff cells that had been preactivated for 3 days with 10 μg/mL anti-CD3 mAb, 1 μg/mL anti-CD28 mAb, and 20 U/mL IL-2 were added to the culture of LSK plus OP-9 cells. Transwell membranes with 0.4 μm pores were used to physically separate the T cells from LSK cells, or alternatively supernatants of cultures of activated Teff cells were added to the LSK culture, making up to 10% of the total culture volume. To assess the effects of cytokines, several cytokines such as IL-6, IL-17, IFN-γ, TNF, and GM-CSF (all at 10 ng/mL; all purchased from Peprotech) were added to the LSK culture. To neutralize cytokines in the LSK culture, anti-IL-6 mAb, antiTNF mAb, anti-GM-CSF mAb, or a mixture of all mAbs (all at 50 μg/mL; all purchased from BioLegend) were added to the LSK culture.
ELISA BM cells were stimulated with 20 ng/mL PMA and 1 μM ionomycin (Sigma-Aldrich, St. Louis, MO, USA), and supernatants were collected after 24 h of culture. Cytokine concentrations in the culture supernatants were measured by sandwich ELISAs using BD OptEIATM kits (BD Biosciences) according to the manufacturer’s instructions.
Statistical analyses Data are presented as mean ± SEM. Differences between groups were evaluated by unpaired Student’s t-tests. p Values are reported for statistically significant differences between groups (
