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Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility

Nature volume 496, pages 523–527 (2013)Cite this article

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Abstract

Germinal centres support antibody affinity maturation and memory formation1. Follicular T-helper cells promote proliferation and differentiation of antigen-specific B cells inside the follicle2,3. A genetic deficiency in the inducible co-stimulator (ICOS), a classic CD28 family co-stimulatory molecule highly expressed by follicular T-helper cells, causes profound germinal centre defects4,5, leading to the view that ICOS specifically co-stimulates the follicular T-helper cell differentiation program2,6,7. Here we show that ICOS directly controls follicular recruitment of activated T-helper cells in mice. This effect is independent from ICOS ligand (ICOSL)-mediated co-stimulation provided by antigen-presenting dendritic cells or cognate B cells, and does not rely on Bcl6-mediated programming as an intermediate step. Instead, it requires ICOSL expression by follicular bystander B cells, which do not present cognate antigen to T-helper cells but collectively form an ICOS-engaging field. Dynamic imaging reveals ICOS engagement drives coordinated pseudopod formation and promotes persistent T-cell migration at the border between the T-cell zone and the B-cell follicle in vivo. When follicular bystander B cells cannot express ICOSL, otherwise competent T-helper cells fail to develop into follicular T-helper cells normally, and fail to promote optimal germinal centre responses. These results demonstrate a co-stimulation-independent function of ICOS, uncover a key role for bystander B cells in promoting the development of follicular T-helper cells, and reveal unsuspected sophistication in dynamic T-cell positioning in vivo.

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Figure 1: Follicular recruitment of activated T cells requires ICOS.
Figure 2: ICOS-dependent follicular T-cell recruitment does not rely on co-stimulation or Bcl6.
Figure 3: ICOSL expressed on bystander B cells is required for follicular T-helper cell recruitment.
Figure 4: ICOS-driven persistent T cell motility in vitro and in vivo.

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Acknowledgements

H.Q. is indebted to his late mother J. Wang for inspiration, and thanks Y. Hong for support. H.Q. was a Tsinghua-Bayer Investigator and a Tsinghua-Janssen Investigator. This work was funded in part by the National Natural Science Foundation of China (grant no. 81072464 and 81161120405), the Program for New Century Excellent Talents in University (no. 20091042000), Tsinghua-Yu-Yuan Medical Sciences Fund (no. 20240000585), Tsinghua University Initiative Scientific Research Program (no. 2010Z02150), Institut Mérieux, and the Tsinghua-Peking Center for Life Sciences.

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  1. Jianfu Li

    Present address: Present address: Department of Oral and Maxillofacial Surgery, The Methodist Hospital Research Institute, Houston, Texas 77030, USA.,

Authors and Affiliations

  1. Tsinghua-Peking Center for Life Sciences, Laboratory of Dynamic Immunobiology, School of Medicine, Tsinghua University, Beijing, 100084, China

    Heping Xu, Xuanying Li, Dan Liu, Xu Zhang, Xin Chen, Shiyue Hou, Lixia Peng & Hai Qi

  2. Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, 100084, China

    Jianfu Li

  3. School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Chenguang Xu & Wanli Liu

  4. Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing, 100021, China

    Lianfeng Zhang

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Contributions

H.X. conducted most of the experiments. H.Q. conceptualized the study, designed the experiments with input from all authors, and wrote the paper. All authors contributed collectively to interpreting data.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16, Supplementary Note 1 and Supplementary References. (PDF 9604 kb)

Spontaneous actin dynamics of T cells-I.

After in vitro activation and retroviral transduction with the LifeAct-mRuby F-actin reporter, T cells were imaged by TIRF microscopy on the isotype antibody coated lipid bilayer at 1 frame per second. Also see corresponding time-lapse images in the left panel of Fig. 4a. (MOV 18205 kb)

Spontaneous actin dynamics of T cells-II.

Another example of TIRF-imaged T cells under the isotype control condition. Data acquired as in Movie S1 at 1 frame per second. (MOV 20603 kb)

An isotropic ICOS-triggering field promotes coordinated actin dynamics, pseudopod extension, and persistent motility of T cells-I.

An example of TIRF-imaged T cells on the anti-ICOS antibody-coated lipid bilayer. Data acquired as in Movie S1 at 1 frame per second. Also see corresponding time-lapse images in the right panel of Fig. 4a. (MOV 15953 kb)

An isotropic ICOS-triggering field promotes coordinated actin dynamics, pseudopod extension, and persistent motility of T cells-II.

Another example of TIRF-imaged T cells on the anti-ICOS antibody-coated lipid bilayer. Data acquired as in Movie S1 at 1 frame per second. Note: in the middle of the image sequence, the cell that started from the upper left briefly lifted its leading edge off the TIRF plane and touched down again toward the end. (MOV 17135 kb)

The motility-promoting effect of ICOS depends on PI3K signaling.

TIRF-imaged T cells on the anti-ICOS antibody-coated lipid bilayer. The cells were treated with p110δ-specific PI3K inhibitor CAL-101. The matching untreated control is in Movie S6 at 1 frame per second. (MOV 9264 kb)

Matching control for Movie S5 and S7.

TIRFimaged T cells on the anti-ICOS antibody-coated lipid bilayer at 1 frame per second. (MOV 14609 kb)

CD28 triggering does not drive persistent motility.

TIRF-imaged T cells on the anti-CD28 antibody-coated lipid bilayer. Note that the T cell did not displace much despite somewhat increased membrane dynamics and polarization. The matching anti-ICOS bilayer control is in Movie S6 at 1 frame per second. (MOV 19345 kb)

Highly coordinated pseudopod dynamics of T cells migrating at the T-B border region in the lymph node.

Three days after being activated in the draining lymph node by s.c. injected OVA323-pulsed DCs, GFP- (green) and dsRed-expressing (red) OT-2 T cells at the T-B border were visualized by 2-photon intravital microscopy. The T-B border region was identified according to follicular B cells and intermixed GFP-expressing T cells (see Methods Summary and Supplementary Fig. 9 for details). The image sequence was acquired at 1 frame per 10 seconds and played back twice in this movie. In the second run, arrowheads highlight pseudopod extensions from T cells. Also see corresponding time-lapse images in Supplementary Fig. 10. Scale bar = 20μm. (MOV 7215 kb)

Icos-/- T cells are impaired in pseudopod dynamics and persistent motility at the T-B border in the lymph node.

GFPexpressing Icos+/+ (green) and dsRed-expressing Icos-/- (red) OT-2 T cells were imaged at the same T-B border. The image sequence was played twice (version A and B). A, regular 20 frames per second replay. B, the depolarized state of both Icos+/+ (4 out of 81, ~5%) and Icos-/- T cells (12 out of 51, ~24%) is marked on 6 equally spaced time frames, exactly as used in the quantitation procedure described in the method. Acquired at 1 frame per 10 seconds. Scale bar = 20μm. (MOV 6336 kb)

The second example for different mobile behaviors of Icos-/- and Icos+/+ T cells at the same T-B border.

Acquired at 1 frame per 10 seconds. Depolarized state: 6% in Icos+/+ vs. 29% in Icos-/-. Scale bar = 20μm. (MOV 2611 kb)

The third example for different mobile behaviors of Icos-/- and Icos+/+ T cells at the same T-B border.

Acquired at 1 frame per 10 seconds. Depolarized state: 8% in Icos+/+ vs. 24% in Icos-/-. Scale bar = 20μm. (MOV 1852 kb)

CD28 triggering does not drive persistent motility.

Comparably impaired pseudopod dynamics and reduced motility of Icos+/+ and Icos-/- T cells at the T-B border when bystander B cells do not express ICOSL. GFP-expressing Icos+/+ (green) and dsRed-expressing Icos-/- (red) OT-2 T cells were imaged at the same T-B border in μMT:Icosl-/- bone-marrow chimera. Note the rounder shape and pausing behaviors of both types of cells. Acquired at 1 frame per 10 seconds. Scale bar = 20μm. (MOV 1816 kb)

Normal naïve T cell motility in the T cell zone does not require ICOS.

Icos+/+ (blue) and Icos-/- (green) naïve CD4 T cells were transferred into naïve B6 mice and imaged in the T cell zone 24-48 hours after transfer. See Supplementary Fig. 2 for typical ICOS expression patterns on naïve T cells. Acquired at 1 frame per 10 seconds. Scale bar = 20μm. (MOV 2324 kb)

Motility of activated T cells in the T cell zone is not reduced in the absence of ICOS.

Icos+/+ (blue) and Icos-/- (green) CD4 T cells were activated in vitro, transferred into naïve B6 mice, and then imaged in the T cell zone 24-48 hours later. See Supplementary Fig. 2 for typical ICOS expression patterns on these activated T cells. Acquired at 1 frame per 10 seconds. Scale bar = 20μm. (MOV 3267 kb)

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Xu, H., Li, X., Liu, D. et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature 496, 523–527 (2013). https://doi.org/10.1038/nature12058

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