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Lymph node stromal cells constrain immunity via MHC class II self-antigen presentation.

Baptista AP, Roozendaal R, Reijmers RM, Koning JJ, Unger WW, Greuter M, Keuning ED, Molenaar R, Goverse G, Sneeboer MM, den Haan JM, Boes M, Mebius RE - Elife (2014)

Bottom Line: In this study, we show that MHC-II expression on lymph node stromal cells is additionally required for homeostatic maintenance of regulatory T cells (Tregs) and maintenance of immune quiescence.MHC-II self-antigen presentation by lymph node stromal cells allowed the non-proliferative maintenance of antigen-specific Tregs and constrained antigen-specific immunity.Altogether, our results reveal a novel mechanism by which lymph node stromal cells regulate peripheral immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands.

ABSTRACT
Non-hematopoietic lymph node stromal cells shape immunity by inducing MHC-I-dependent deletion of self-reactive CD8(+) T cells and MHC-II-dependent anergy of CD4(+) T cells. In this study, we show that MHC-II expression on lymph node stromal cells is additionally required for homeostatic maintenance of regulatory T cells (Tregs) and maintenance of immune quiescence. In the absence of MHC-II expression in lymph node transplants, i.e. on lymph node stromal cells, CD4(+) as well as CD8(+) T cells became activated, ultimately resulting in transplant rejection. MHC-II self-antigen presentation by lymph node stromal cells allowed the non-proliferative maintenance of antigen-specific Tregs and constrained antigen-specific immunity. Altogether, our results reveal a novel mechanism by which lymph node stromal cells regulate peripheral immunity.

No MeSH data available.


Related in: MedlinePlus

Endogenous OVA presentation by lymph node stromal cells does not affect CD4+Foxp3− conventional T cells.MACS-sorted CD4+ wild-type or OT-II transgenic cells were cultured together with in vitro-generated stromal cell lines of K14-mOVA origin in the absence or presence of the MHC-II blocking antibody M5/114. After 72 hr of co-culture, OT-II cells were characterized by flow cytometry. The number of CD4+Fox3− conventional T cells in the beginning and at the end of culture is shown. Data represent mean ± SEM. n = 3 for wild-type CD4+ T cells; n = 8 for OT-II cells; and n = 3 for OT-II cells + M5/114.DOI:http://dx.doi.org/10.7554/eLife.04433.017
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fig4s3: Endogenous OVA presentation by lymph node stromal cells does not affect CD4+Foxp3− conventional T cells.MACS-sorted CD4+ wild-type or OT-II transgenic cells were cultured together with in vitro-generated stromal cell lines of K14-mOVA origin in the absence or presence of the MHC-II blocking antibody M5/114. After 72 hr of co-culture, OT-II cells were characterized by flow cytometry. The number of CD4+Fox3− conventional T cells in the beginning and at the end of culture is shown. Data represent mean ± SEM. n = 3 for wild-type CD4+ T cells; n = 8 for OT-II cells; and n = 3 for OT-II cells + M5/114.DOI:http://dx.doi.org/10.7554/eLife.04433.017

Mentions: The observation that MHC-II expression on lymph node stromal cells impacted on the peripheral maintenance of Tregs implied that cognate ligands for the T cell receptor (TCR) of Tregs are expressed and presented by the lymph node stromal cell compartment. In support of such hypothesis, previous research has reported expression of several peripheral tissue-restricted antigens (PTAs) in lymph node stromal cells (Nichols et al., 2007; Cohen et al., 2010; Fletcher et al., 2010). To assess whether lymph node stromal cells were able to present endogenous antigens in the context of MHC-II molecules, we used K14-mOVA transgenic mice, in which ovalbumin (OVA) expression is driven by the human keratin 14 promoter (Bianchi et al., 2009). In these mice, OVA is expressed in the skin and thymus (Bianchi et al., 2009) as well as in the 3 major lymph node stromal cell subsets (Figure 4A). As all primary lymph node stromal cell cultures established contained large amounts of contaminating hematopoietic cells (data not shown), which precluded our in vitro antigen presentation assays, we generated distinct lymph node stromal cell lines by long-term in vitro culture of lymph node single cell suspensions of K14-mOVA mice on collagen matrixes. Using this approach, we generated one cell line (K14-mOVAneg), resembling FRCs, which did not express detectable OVA mRNA transcripts and was thus used as a control cell line, and another cell line (K14-mOVApos), resembling LECs, which expressed OVA transcripts abundantly (Figure 4B and Figure 4—figure supplement 1). Co-culture of these cell lines with OVA-specific CD8+ OT-I and CD4+ OT-II T cells revealed that OVA-derived peptides could be presented by lymph node stromal cells in MHC-I as well as MHC-II molecules, as both OT-I and OT-II T cells showed increased CD25 expression when cultured with K14-mOVApos but not with K14-mOVAneg cells (Figure 4—figure supplement 2). Neither lymph node stromal cell line induced OT-I or OT-II T cell proliferation, however (Figure 4—figure supplement 2). To directly address whether self-antigen presentation by lymph node stromal cells in the context of MHC-II molecules influenced Tregs, we repeated our co-culture experiments and stained CD4+ T cells for Foxp3 and Helios. As compared to culture in medium alone, co-culture of CD4+ T cells with either cell line increased the survival of Foxp3+ cells irrespective of their TCR specificity (Figure 4C), which is suggestive of the production of T cell survival factors by both cell lines (Link et al., 2007). More importantly, K14-mOVApos cells significantly increased the recovery of Foxp3+ OT-II T cells as compared to the K14-mOVAneg cell line (Figure 4C). This effect was not apparent in co-cultures with wild-type CD4+ T cells and could be blocked by the addition of the MHC-II blocking antibody M5/114 (Figure 4C), indicating that the presentation of OVA-derived peptides in the context of MHC-II molecules by the K14-mOVApos cell line was the driver of increased Foxp3+ OT-II T cell survival in our assays. Of significance, CD4+Foxp3− conventional OT-II T cells, in the exact same conditions, did not show similar behavior (Figure 4—figure supplement 3). Overall, our data suggest that MHC-II-mediated self-antigen presentation by lymph node stromal cells drives CD4+Foxp3+ Treg maintenance.10.7554/eLife.04433.014Figure 4.Endogenous OVA presentation by lymph node stromal cells promotes Treg maintenance in vitro.


Lymph node stromal cells constrain immunity via MHC class II self-antigen presentation.

Baptista AP, Roozendaal R, Reijmers RM, Koning JJ, Unger WW, Greuter M, Keuning ED, Molenaar R, Goverse G, Sneeboer MM, den Haan JM, Boes M, Mebius RE - Elife (2014)

Endogenous OVA presentation by lymph node stromal cells does not affect CD4+Foxp3− conventional T cells.MACS-sorted CD4+ wild-type or OT-II transgenic cells were cultured together with in vitro-generated stromal cell lines of K14-mOVA origin in the absence or presence of the MHC-II blocking antibody M5/114. After 72 hr of co-culture, OT-II cells were characterized by flow cytometry. The number of CD4+Fox3− conventional T cells in the beginning and at the end of culture is shown. Data represent mean ± SEM. n = 3 for wild-type CD4+ T cells; n = 8 for OT-II cells; and n = 3 for OT-II cells + M5/114.DOI:http://dx.doi.org/10.7554/eLife.04433.017
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4270074&req=5

fig4s3: Endogenous OVA presentation by lymph node stromal cells does not affect CD4+Foxp3− conventional T cells.MACS-sorted CD4+ wild-type or OT-II transgenic cells were cultured together with in vitro-generated stromal cell lines of K14-mOVA origin in the absence or presence of the MHC-II blocking antibody M5/114. After 72 hr of co-culture, OT-II cells were characterized by flow cytometry. The number of CD4+Fox3− conventional T cells in the beginning and at the end of culture is shown. Data represent mean ± SEM. n = 3 for wild-type CD4+ T cells; n = 8 for OT-II cells; and n = 3 for OT-II cells + M5/114.DOI:http://dx.doi.org/10.7554/eLife.04433.017
Mentions: The observation that MHC-II expression on lymph node stromal cells impacted on the peripheral maintenance of Tregs implied that cognate ligands for the T cell receptor (TCR) of Tregs are expressed and presented by the lymph node stromal cell compartment. In support of such hypothesis, previous research has reported expression of several peripheral tissue-restricted antigens (PTAs) in lymph node stromal cells (Nichols et al., 2007; Cohen et al., 2010; Fletcher et al., 2010). To assess whether lymph node stromal cells were able to present endogenous antigens in the context of MHC-II molecules, we used K14-mOVA transgenic mice, in which ovalbumin (OVA) expression is driven by the human keratin 14 promoter (Bianchi et al., 2009). In these mice, OVA is expressed in the skin and thymus (Bianchi et al., 2009) as well as in the 3 major lymph node stromal cell subsets (Figure 4A). As all primary lymph node stromal cell cultures established contained large amounts of contaminating hematopoietic cells (data not shown), which precluded our in vitro antigen presentation assays, we generated distinct lymph node stromal cell lines by long-term in vitro culture of lymph node single cell suspensions of K14-mOVA mice on collagen matrixes. Using this approach, we generated one cell line (K14-mOVAneg), resembling FRCs, which did not express detectable OVA mRNA transcripts and was thus used as a control cell line, and another cell line (K14-mOVApos), resembling LECs, which expressed OVA transcripts abundantly (Figure 4B and Figure 4—figure supplement 1). Co-culture of these cell lines with OVA-specific CD8+ OT-I and CD4+ OT-II T cells revealed that OVA-derived peptides could be presented by lymph node stromal cells in MHC-I as well as MHC-II molecules, as both OT-I and OT-II T cells showed increased CD25 expression when cultured with K14-mOVApos but not with K14-mOVAneg cells (Figure 4—figure supplement 2). Neither lymph node stromal cell line induced OT-I or OT-II T cell proliferation, however (Figure 4—figure supplement 2). To directly address whether self-antigen presentation by lymph node stromal cells in the context of MHC-II molecules influenced Tregs, we repeated our co-culture experiments and stained CD4+ T cells for Foxp3 and Helios. As compared to culture in medium alone, co-culture of CD4+ T cells with either cell line increased the survival of Foxp3+ cells irrespective of their TCR specificity (Figure 4C), which is suggestive of the production of T cell survival factors by both cell lines (Link et al., 2007). More importantly, K14-mOVApos cells significantly increased the recovery of Foxp3+ OT-II T cells as compared to the K14-mOVAneg cell line (Figure 4C). This effect was not apparent in co-cultures with wild-type CD4+ T cells and could be blocked by the addition of the MHC-II blocking antibody M5/114 (Figure 4C), indicating that the presentation of OVA-derived peptides in the context of MHC-II molecules by the K14-mOVApos cell line was the driver of increased Foxp3+ OT-II T cell survival in our assays. Of significance, CD4+Foxp3− conventional OT-II T cells, in the exact same conditions, did not show similar behavior (Figure 4—figure supplement 3). Overall, our data suggest that MHC-II-mediated self-antigen presentation by lymph node stromal cells drives CD4+Foxp3+ Treg maintenance.10.7554/eLife.04433.014Figure 4.Endogenous OVA presentation by lymph node stromal cells promotes Treg maintenance in vitro.

Bottom Line: In this study, we show that MHC-II expression on lymph node stromal cells is additionally required for homeostatic maintenance of regulatory T cells (Tregs) and maintenance of immune quiescence.MHC-II self-antigen presentation by lymph node stromal cells allowed the non-proliferative maintenance of antigen-specific Tregs and constrained antigen-specific immunity.Altogether, our results reveal a novel mechanism by which lymph node stromal cells regulate peripheral immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands.

ABSTRACT
Non-hematopoietic lymph node stromal cells shape immunity by inducing MHC-I-dependent deletion of self-reactive CD8(+) T cells and MHC-II-dependent anergy of CD4(+) T cells. In this study, we show that MHC-II expression on lymph node stromal cells is additionally required for homeostatic maintenance of regulatory T cells (Tregs) and maintenance of immune quiescence. In the absence of MHC-II expression in lymph node transplants, i.e. on lymph node stromal cells, CD4(+) as well as CD8(+) T cells became activated, ultimately resulting in transplant rejection. MHC-II self-antigen presentation by lymph node stromal cells allowed the non-proliferative maintenance of antigen-specific Tregs and constrained antigen-specific immunity. Altogether, our results reveal a novel mechanism by which lymph node stromal cells regulate peripheral immunity.

No MeSH data available.


Related in: MedlinePlus