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. 2016 Jul 5;113(27):7608-13.
doi: 10.1073/pnas.1521846113. Epub 2016 Jun 21.

Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival

Mohammad Nizam Uddin  1 , Dil Afroz Sultana  1 , Kyle J Lorentsen  1 , Jonathan J Cho  1 , Mariana E Kirst  1 , Mark L Brantly  1 , Danielle Califano  2 , Derek B Sant'Angelo  3 , Dorina Avram  4
Affiliations

Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival

Mohammad Nizam Uddin et al. Proc Natl Acad Sci U S A. .

Abstract

Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

Keywords: iNKT cell program; iNKT1 effector cells; iNKT17 effector cells; iNKT2 effector cells; transcription factor Bcl11b.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Bcl11b is specifically removed from iNKT cells in Bcl11bF/F/PLZF-Cre mice and not from CD4+ T cells or innate lymphoid type 2 cells (ILC2s). (A and B) Frequencies of Bcl11b+ PBS-57/CD1d+ T cells and PBS-57/CD1d− CD4+ T cells in thymus (A) and spleen (B) of Bcl11bF/F/PLZF-Cre and Bcl11bF/F mice evaluated by FACS. (C) Frequencies of Bcl11b+ iNKT cells in the four thymic developmental stages from Bcl11bF/F/PLZF-Cre and Bcl11bF/F mice. (D and E) Frequencies of DP, CD4, and CD8 SP thymocytes (D), and CD4+ and CD8+ T cells in spleen (E) of Bcl11bF/F/PLZF-Cre and WT control mice. (F) Bcl11b in ILC2s from mesenteric lymph nodes and lung of Bcl11bF/F/PLZF-Cre and WT mice. (G) St2 and Rorγt in lung Lin− CD90+CD127+CD25+ innate lymphoid cells of Bcl11bF/F/PLZF-Cre and WT mice. Data are representative of five pairs of mice (A and B), four pairs of mice (C), and three pairs of mice (D–G).
Fig. 1.
Fig. 1.
Bcl11b's deficiency in iNKT cells causes a major reduction in thymic and peripheral iNKT cells associated with increased apoptosis. (A and B) Frequencies (A) and absolute numbers (B) of iNKT cells from Bcl11bF/F/PLZF-Cre (KO) and WT mice in thymus, spleen, and liver. The gated population in A shows percentages of CD1d-PBS-57+cells. (C) Frequencies of Annexin V+ iNKT cells from Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice in thymus and spleen. Data are representative of several independent experiments with 10 pairs of mice (A and B) and 5 pairs of mice (C). P values determined by unpaired two-tailed Student’s t test are indicated. Means ± SEM.
Fig. S2.
Fig. S2.
Frequencies of iNKT cells in thymus, spleen, and liver of Bcl11bF/F/PLZF-Cre mice, survival of iNKT cells and levels of CD1d-PBS-57 bound by Bcl11b−/− iNKT cells. (A) Quantification of percentages of iNKT cells from Bcl11bF/F/PLZF-Cre and WT mice in thymus, spleen, and liver. (B) Mean fluorescent intensity (MFI) of CD1d-PBS-57 bound to iNKT cells from thymus, spleen, and liver of Bcl11bF/F/PLZF-Cre and Bcl11bF/F mice. Data are representative of eight pairs of mice (A) and five pairs of mice (B). P values are indicated. Means ± SEM. (C) Frequencies of Annexin V+ iNKT cells from Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice in thymus and spleen. Data are representative of five pairs of mice.
Fig. 2.
Fig. 2.
Bcl11b's removal in iNKT cells causes developmental alterations. (A) Frequencies of iNKT cells from Bcl11bF/F/PLZF-Cre and WT mice in the four developmental stages (Left) based on surface CD24, CD44, and NK1.1 within the CD1d-PBS-57+ cells. (B and C) Quantification of percentages (B) and absolute numbers (C) of iNKT cells within the four developmental stages. Data are representative of several independent experiments with 10 pairs of mice. P values are indicated. Means ± SEM.
Fig. S3.
Fig. S3.
Ki67 levels in developmental stages 1 and 2. Frequencies of Ki67+ iNKT cells in thymic developmental stages 1 and 2 of Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice. Data are representative of five pairs of mice. Means ± SEM.
Fig. 3.
Fig. 3.
Bcl11b-deficient iNKT cells show altered expression of genes critical for effector iNKT subsets and survival. Scatterplot of log2 expression values for mRNAs isolated from sorted iNKT cells of Bcl11bF/F/PLZF-CreVα14 Tg (KO) versus Vα14 Tg control mice (WT) treated with α-GalCer. Data are representative of two independent arrays. Relevant genes are listed. The heat map shows relevant genes off the scale in scatterplot.
Fig. 4.
Fig. 4.
iNKT1 and iNKT2 subset cell numbers are reduced in thymus and spleen of Bcl11bF/F/PLZF-Cre mice. (A–D) Frequencies (A and B) and absolute numbers (C and D) of effector iNKT subsets in thymus and spleen, identified as iNKT1 (TbethiPLZFlo), iNKT2 (TbetloPLZFhi), and iNKT17 (RorγthiPLZFlo) on gated CD1d-PBS-57+ cells from Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice. Data are representative of 8–10 pairs of mice. P values are indicated. Means ± SEM.
Fig. S4.
Fig. S4.
Bcl11b is expressed in all effector iNKT subsets and its depletion alters the frequencies of iNKT subsets. (A) Frequencies of Bcl11b+ cells in the thymic and splenic iNKT subsets defined as in Fig. 4. (B and C) Percentages of effector iNKT subsets in thymus (B) and spleen (C) of Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice. Data are representative of five pairs of mice. P values are indicated. Means ± SEM.
Fig. 5.
Fig. 5.
Bcl11b-deficient iNKT cells are functionally altered. (A–F) Frequencies (A–D) and absolute numbers (E and F) of IL-4+, IFNγ+, and IL-17+ iNKT cells within the indicated effector subsets, iNKT1, iNKT2, and iNKT17, defined as in Fig. 4 A and B. Data are representative of several experiments with eight pairs of mice. P values are indicated. Means ± SEM.
Fig. S5.
Fig. S5.
Bcl11b-deficient iNKT cells show altered cytokine production. Frequencies of IL-4–, IFNγ- (Top), and IL-4–, and IL-17 (Bottom)-producing iNKT cells in thymus (A) and spleen (B) from Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice. For evaluation of cytokine production in thymus, total thymocytes were collected and treated in vitro with α-galactosylceramide (α-GalCer) for 72 h (A). For evaluation of splenic iNKT cells, mice were injected i.p. with α-GalCer and killed 4 h later (B). Data are representative of six pairs of mice.
Fig. 6.
Fig. 6.
Bcl11b-deficient iNKT cells, including iNKT1 and iNKT2 effector subsets up-regulate iNKT17 cell markers. Frequencies of IL-23R+, Nrp1+, NKp46+, and Granzyme B+ in total iNKT cells and in effector iNKT subsets of Bcl11bF/F/PLZF-Cre and Bcl11bF/F mice in thymus (A) and spleen (B). iNKT1, iNKT2, and iNKT17 are defined as in Fig. 4 A and B. Data are representative of three independent experiments.
Fig. S6.
Fig. S6.
Lef1 and Gata3 are not reduced in Bcl11b−/− iNKT cells belonging to the iNKT2 subset and CD8 is not up-regulated on Bcl11b-deficient iNKT cells. (A) Frequencies of Lef1+ and Gata3+ total iNKT and iNKT2 cells in thymus of Bcl11bF/F/PLZF-Cre and Bcl11bF/F mice evaluated by FACS. (B) Frequencies of thymic CD8+ iNKT cells from Bcl11bF/F/PLZF-Cre (KO) and Bcl11bF/F (WT) mice. Data are representative of of three pairs of mice.
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