T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse - PubMed (original) (raw)

T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse

Amy E Moran et al. J Exp Med. 2011.

Abstract

The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (T(reg) cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although T(reg) cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that T(reg) cell progenitors compete for recognition of rare stimulatory TCR self-ligands.

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Figures

Figure 1.

A Nur77GFP BAC transgenic mouse expresses GFP upon TCR activation. (A) A GFP-Cre fusion protein was inserted at the start site of the Nr4a1 (Nur77) gene of a BAC construct and used to generate B6 or B6.SJLF1 transgenic lines. (B) GFP was highly expressed in a subset of myeloid cells of the spleen but not lymph node. (C) T and B lymphocytes expressed a low level of GFP. Three founder lines showed similar cell-specific patterns of GFP expression, but higher levels were observed in the B6-820 line (n = 5 mice). (D) GFP was up-regulated in T cells 12 h after anti-CD3 injection in vivo or in B cells after 3 h of anti-IgM treatment in vitro (n = 4 mice and three experiments).

Figure 2.

The level of GFP expression reflects TCR signal strength and is transient. (A) OT-I/Tapo/Nur77GFP thymocytes were co-cultured for 3 h with B6 splenocytes pulsed with the peptide SIINFEKL (OVAp) or the indicated altered peptide ligands, listed in order of decreasing potency. The MFI of GFP was normalized to the level observed with OVAp stimulation (n = 5). cntl, control. (B) Splenocytes from OT-I/Nur77GFP mice were cultured with the indicated peptides for various lengths of time. The data represent the mean normalized GFP levels from six different experiments of at least six mice. Error bars indicate standard deviation.

Figure 3.

GFP expression is induced during positive selection. (A) Flow cytometric analysis of GFP in total thymocytes (left). Dot plots (right) show CD4 and CD8 expression on total or GFP-positive thymocytes from Nur77GFP mice. (B) The GFP+ DP population was enriched for CD69+TCR-β+ cells (dot plot). CD69+TCR-βhi (postselection) DP thymocytes expressed higher levels of GFP compared with CD69−TCR-βlo (preselection) DP thymocytes (histogram overlay). (C) GFP expression of DP thymocytes from WT or Tap-deficient mice. NTg, nontransgenic. (D) Immunofluorescence analysis of GFP in the Nur77GFP thymus, with the cortical region defined by staining for the β5t proteasome subunit. Data are representative of >10 mice from at least three independent experiments. Bars, 100 µm.

Figure 4.

GFP expression is maintained in the steady-state by tonic MHC signals. (A) Analysis of GFP levels in mature CD4 and CD8 SP thymocytes (left; defined as HSAloQa2hi) or naive phenotype CD4 and CD8 lymph node T cells (right; defined as CD44loCD69−CD25−). Data are representative of >10 mice. (B) 1–2 × 106 polyclonal Nur77GFP CD4 T cells were transferred into B6 or I-Ab–deficient (MHC IIo) recipients and analyzed 6 or 9 d later. Bar graph shows the mean GFP level on cells adoptively transferred into I-Ab–deficient recipients normalized to the level on CD4 T cells in B6 recipients. Data are representative of 11 mice from four independent experiments. Error bars indicate standard deviation. NTg, nontransgenic; Tg, transgenic.

Figure 5.

Thymocytes undergoing negative selection express higher levels of GFP compared with those undergoing positive selection. OT-I/Nur77GFP mice or OT-I/Bimo/Nur77GFP mice were generated and used as bone marrow donors. 5–10 × 106 bone marrow cells were injected into lethally irradiated B6 or RIP-mOVA recipients. (A) Expression of CD4 and CD8 on thymocytes from the indicated chimeric mice. (B) GFP expression on Vα2+ CD8SP from the indicated chimeric mice. Representative data are from five experiments with more than five mice. NTg, nontransgenic.

Figure 6.

Induction of GFP is TCR specific. (A and B) Nur77GFP mice were injected i.v. with pI:pC (A) or LPS (B). After 6 h, cells were analyzed for GFP (left) and CD69 (right) expression. (C) 5 × 106 OT-I/Nur77GFP lymph node cells were adoptively transferred into B6 recipients and infected with L. monocytogenes expressing the OVAp (LM-OVA) or not (LM). GFP expression on Vα2+CD8+ transferred cells was evaluated after 12 h. Histograms show representative data from three independent experiments with at least three mice.

Figure 7.

Treg cells express higher levels of GFP compared with Tconv cells. Lymphoid organs from Nur77GFP mice were analyzed by flow cytometry. Treg cells were defined as CD4SP CD25+Foxp3+. Treg cell progenitors were CD4SP CD25+CD122hiFoxp3−. Data are representative of more than eight mice from eight experiments. NTg, nontransgenic.

Figure 8.

Stat5 signaling does not increase GFP in Treg or Tconv cells. Stat5b-CA/Nur77GFP and Nur77GFP mice were generated. (A) Increased frequency of CD4+Foxp3+ Treg cells in Stat5b-CA/Nur77GFP mice in both the thymus and periphery. (B) Histogram overlays of CD4+Foxp3+ cells of Stat5b-CA/Nur77GFP and Nur77GFP control thymocytes (left) and lymphocytes (right). (C) Histogram overlays of CD4+Foxp3− CD4 T cells from Stat5b-CA/Nur77GFP and Nur77GFP mice. Data are representative of three experiments with four mice per group. NTg, nontransgenic.

Figure 9.

Treg cells compete for strong TCR ligands during development. G113/Rag1o/Nur77GFP (CD45.2+) and WT (CD45.1+) bone marrow were mixed at various ratios and used to reconstitute lethally irradiated recipients (CD45.1+). Chimerism was determined by analyzing the G113+ T cell fraction from lymphoid tissue. 8 wk after transplant, lymphoid organs were harvested and analyzed by flow cytometry. (A) Dot plots on the far left show percent chimerism from select animals ranging from 97 to 0.032% G113 donor–derived T cells. Histograms on the right show GFP after gating on CD45.2+Vα2+Vβ6+CD4SP that were either CD25 negative or positive. (B) Similar mixed bone marrow chimeras were set up with OT-II/Nur77GFP bone marrow. Graph shows the GFP MFI on Vβ6+ CD4SP thymocytes (for G113) or Vβ5+ CD4SP (for OT-II). Data are representative of three experiments with more than five mice. n.d., not determined. NTg, nontransgenic.

Figure 10.

NKT cells express higher levels of GFP immediately after selection but do not maintain it in the periphery. (A) Histograms show the level of GFP on conventional CD4SP or CD1d/α-GalCer tetramer–binding iNKT cells in the thymus or spleen of Nur77GFP mice. Data are representative of more than five mice in at least three independent experiments. (B) GFP levels on splenic iNKT cells 6 h after injection with α-GalCer or solvent control. (C) CD1d/α-GalCer tetramer–binding iNKT cells were enriched from adult thymus using magnetic beads. Dot plots show the gating strategy used to identify subsets of thymic iNKT cells. (D) GFP levels on various staged iNKT cell populations compared with conventional CD4SP thymocytes. Data are representative of four experiments and more than nine mice. NTg, nontransgenic.

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T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse - PubMed (original) (raw)