Migration-induced cell shattering due to DOCK8 deficiency causes a type 2–biased helper T cell response

Abstract

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8−/− mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8−/−CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1β that drives granulocyte−macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1β, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

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Fig. 1: Dock8 deficiency leads to a type 2 immunity bias upon infection.
Fig. 2: Dock8 deficiency results in a type 2–biased CD4+ T cell response to non-TH2 stimuli and is associated with greater GM-CSF production.
Fig. 3: Selective loss of Dock8 in CX3CR1+ mononuclear phagocytes recapitulates the type 2 immunity bias.
Fig. 4: Mononuclear phagocytes lacking Dock8 are highly sensitive to migration-induced shattering, caspase activation and death.
Fig. 5: Increased IL-1β is a necessary but not sufficient signal in the greater GM-CSF and type 2 cytokine production by CD4+ T cells in Dock8 deficiency.
Fig. 6: GM-CSF production by CD4+ T cells plays a role in the type 2 bias, amplifying IL-1β production.
Fig. 7: Cell death provides a necessary and sufficient type 2–inducing signal.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on request. Source data are provided with this paper.

Code availability

Custom MATLAB code to quantify cell shape parameters is available via public GitHub repository at https://github.com/Wiseman-Lab/image-segmentation.

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Acknowledgements

We thank P. D’Arcy, G. Perreault and animal facility staff at McGill for their excellent care of our animal colony; K. Mayer-Barber (NIH) for technical advice on myeloid cell stimulations; N. van Panhuys (Sidra Medical and Research Center, Qatar) for helpful discussions; J. Lee (Mayo Clinic) for providing Eo-cre mice; J. Burkhardt (U Penn) for the LifeAct-GFP mice; and D. Malo (McGill) for sharing the LysM-cre mice. Influenza A virus was provided by S. Vidal at The Centre for Phenogenomics McGill. A stock of C.neoformans H99 was shared by K. Kwon-Chung (NIH). We thank H. Matthews and P. Angelus for clinical support at NIH. R. Germain (NIH), S. Schwab (NYU), M. Richer (McGill), H. Melichar (University of Montreal), S. Gruenheid (McGill) and Mandl laboratory members provided critical feedback and advice on previous manuscript drafts. We thank the McGill University Cell Vision Core Facility for flow cytometry and single-cell analysis, the Life Sciences Complex Advanced BioImaging Facility and the Goodman Cancer Research Centre Histology Core Facility. C. Schneider was funded by the Max E. Binz Fellowship, McGill University Faculty of Medicine; C. Shen and P.A. were funded by a Fonds de recherche Santé Quebec fellowship; and D.R. was funded by a Tomlinson Doctoral Fellowship. J.N.M. is a Canada Research Chair for immune cell dynamics. M.S. is a McGill William Dawson Scholar. This research was supported by a Canadian Institutes of Health Research (CIHR) project grant (201603PJT-364017) and McGill start-up funds to J.N.M.; an NSERC Discovery Grant to P.W.W. (2017-05005), two CIHR project grants and an NSERC Discovery Grant to M.S.; a CIHR grant (MOP-130579) to I.L.K.; and by the Intramural Research Programme of the NIAID, NIH (D.L.B., A.F.F. and H.C.S.).

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J.N.M. conceived the project. C. Schneider and J.N.M. designed the research. C. Schneider performed most of the experiments with critical help from C. Shen, B.F., K.D.K., P.A. and D.R. A.A.G. contributed some of the BMDC and T cell collagen matrix movies and associated analyses of cell shape with input from P.W.W. C. Shen contributed the CFA experiments, with input from I.L.K., and higher-resolution images and movies of migrating BMDCs and T cells. T.D. performed the immunoblots for caspase activation with input from M.S. Data were analyzed by J.N.M., C. Schneider, A.A.G., C. Shen, M.S. and P.W.W. At NIH, A.F.F. and H.C.S. provided human samples that were processed and analyzed by H.J. Critical reagents and intellectual input were provided by I.L.K., M.S., D.L.B, Q.Z. and H.C.S. The manuscript was written by J.N.M. and C. Schneider, with all authors contributing and providing feedback.

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Correspondence to Judith N. Mandl.

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Editor recognition statement Jamie D. K. Wilson and Ioana Visan were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Dock8-/- mice survive significantly longer than WT mice after C. neoformans infection despite fungal dissemination.

a, b, Wild type (WT) or Dock8-deficient (KO) mice were infected with C. neoformans and cells harvested from lung 20 days p.i. Production of cytokines was assessed by intracellular flow cytometry staining after ex vivo restimulation and total number of activated CD4 + T cells making IL-4, IL-5, and IL-13 (a), or making any Th2 cytokine (IL-4 or IL-5 or IL-13) (b), shown from 4 independent experiments; n = 29 WT and 18 KO. c,d, Mice were infected with 50,000 colony forming units (CFU) of C. neoformans and euthanized when weight loss >20% of initial body weight, between 28–35 (WT) or 30–45 (KO) days p.i. CFU in spleen and brain at euthanasia (c), and Kaplan-Meyer survival plots (d). Data from 2 independent experiments; n = 11 WT and 16 KO. e–g, Mice were infected as in (a, b) and on day 20 p.i. total CD4 + T cell counts in the non-draining inguinal lymph node (ndLN) (e), and number of activated (CD44-hi) CD4 + T cells in draining mediastinal lymph node (dLN) and lung (f). Data from 3–4 independent experiments; n = 15 per group. Percent CD4 + T cells with an activated (CD44-hi) or naive (CD44-lo) phenotype in lung parenchyma versus lung vasculature as determined by i.v. antibody labeling and analysis by flow cytometry summarized from 2 independent experiments, n = 10 per group (g). In all graphs, error bars represent group medians ± min/max with box boundaries denoting 25th and 75th percentiles, except in (g) mean ± SD shown; each data point is from an individual mouse. Statistical analyses were performed using: two-tailed Kruskal-Wallis ANOVA (a, c); Mantel-Cox log rank test (d); or two-tailed Mann-Whitney test (b, eg). *P < 0.05, **P < 0.01, and ****P < 0.0001.

Extended Data Fig. 2 Dock8 deficiency does not greatly alter CD4 T cell cytokine production in response to helminth infection.

a, Concentration of IL-12p70 in total lung homogenate at 20 days p.i. with C. neoformans. Data from one experiment; n = 5 per group; b–d, Mice were infected intranasally with Influenza A virus and 7 days p.i. number of activated CD4 + T cells (b), and eosinophils (c), quantified in the lung, and viral load measured by RT-qPCR of nonstructural protein (Ns1) mRNA in lung homogenate (d). Data from 2 independent experiments; for b, c, n = 9 per group; for d, n = 5 WT and 9 KO. e, f, Mice were immunized subcutaneously with Complete Freund’s adjuvant (CFA) and OVA peptide. The number of activated CD4 + T cells (e), and eosinophils (f), quantified in the iliac lymph node 7 days later; data from one of 3 independent experiments shown, n = 5 per group. g–i, Mice were orally infected with H. polygyrus, and number of activated (CD44-hi) CD4 + T cells (g), frequency of cytokine-producing activated CD4 + T cells (h), and number of eosinophils (i) measured in the mesenteric lymph nodes 7 days p.i.; data from 2 independent experiments; n = 8 per group. Lines denote group means in a, d; error bars represent group medians ± min/max with box boundaries denoting 25th and 75th percentiles in b, c, e–i. In all graphs each data point is from an individual mouse. All statistical analyses were performed using two-tailed Mann-Whitney tests. *P < 0.05 and **P < 0.01.

Extended Data Fig. 3 The Th2 skew in Dock8-deficiency is intrinsic to the hematopoietic compartment.

a, Chimeras were generated by injection of WT bone marrow into Dock8-/- recipients, which were infected 8 weeks later with C. neoformans in parallel with non-chimeric WT and KO controls. Lung cells were restimulated ex vivo to assess cytokine production by CD4 + T cells. Data from 1 experiment; n = 4 WT, 3 KO, and 7 WT - > KO; lines denote group means. Statistical analyses were performed using Kruskal-Wallis ANOVA. *P < 0.05 and ****P < 0.0001. b, Gating strategy example for lung cells. Cells are gated on live, singlet cells (not shown) and delineated as shown. AMϕ = alveolar macrophages, DCs = dendritic cells, moDCs = monocyte-derived DCs, cDCs = conventional DCs, MNPs = mononuclear phagocytes, monos = monocytes, Mϕ = macrophages.

Extended Data Fig. 4 The Th2 skew in Dock8-deficiency is extrinsic to ILCs, DCs, T cells, neutrophils, and eosinophils.

a, RT-qPCR measure of Dock8 knock-down efficiency in purified T cells, and splenic DCs as percent of WT Dock8 mRNA levels, normalized by GAPDH expression. Data from 1 experiment, n = 3 mice per group. b, c, WT (n = 32), KO (n = 29), or Dock8fl/fl mice crossed to Lck (n = 9), CD11c (n = 10), LysM (n = 10) or eosinophil-specific (n = 9) cre lines were infected with C. neoformans. Total lung eosinophil counts (b), and ex vivo production of GM-CSF and IL-17 by CD4 + T cells assessed by flow cytometry 20 days p.i. (c). Data from 2 independent experiments for each group. d, Dock8-/- mice were depleted of B cells (anti-CD20) the day before infection with C. neoformans and ex vivo production of IL-4, IL-5 or IL-13 by lung CD4 + T cells assessed 16 days p.i. Data from 1 experiment; n = 5 each for WT and KO and n = 4 for treated KO. e–h, Lung ILCs were gated as lineage-, TCRβ-, CD4-, CD8-, CD45 + , Thy1 + , IL-7R + on 4 and 20 days post C. neoformans infection. Representative flow cytometry plots shown in (e) with percent of cells in gate (blue text, top) and percent of total lung (black text, bottom), and data summarized from 2 independent experiments (f; n = 3 WT and 7 KO). Representative flow cytometry plots showing percent of ILCs that are RORγt+ or GATA-3 + (g), and data summarized from 1 representative experiment (h; n = 4 WT and 5 KO). i, RT-qPCR measure of Dock8 knock-down efficiency in purified monocytes (CX3CR1 + ) as a percent of WT Dock8 mRNA levels, normalized by GAPDH expression. Data from 1 experiment, n = 3 mice per group. j, WT, KO, or CX3CR1-Dock8-/- mice were infected with C. neoformans and cytokine−producing lung CD4 + T cells measured 8 days p.i. Data from 3 independent experiments; n = 13 control, 16 KO, and 13 CX3CR1-Dock8-/- mice. Control group includes: WT mice (black circles), corn-oil treated Dock8fl/fl. CX3CR1-ERcre + (white circles), and tamoxifen-treated Dock8fl/fl. CX3CR1-ERcre– (gold circles). For all graphs, each data point is from an individual mouse. Lines denote means in (f); mean ± sem is shown in (a, i); error bars represent group medians ± min/max with box boundaries denoting 25th and 75th percentiles in (b, c, d, h, j). Statistical analyses were performed using: two-tailed Kruskal-Wallis ANOVA (bd, h, j) or Mann-Whitney test (f). *P < 0.05, **P < 0.01, and ****P < 0.0001.

Extended Data Fig. 5 Abnormal morphology of Dock8-/- T cells during migration ex vivo and non-Th2 cytokine production during caspase block or in absence of IL-1R signaling.

a, Quantification of T cell area, perimeter, major and minor axes after 3 h of migration in a 3.6 mg/mL collagen matrix. Data from 4 movies, 2 mice/group (n = 941 WT and n = 1058 KO cells). b, Example confocal images of stretching LifeAct-GFP + T cells from (a). Scale bar is 10μm. c, KO mice were treated with Q-VD-OPh during C. neoformans infection and frequency of activated lung CD4 + T cells making cytokines was assessed on day 8. Data from 2 independent experiments; n = 8 WT, 10 KO, and 10 treated KO mice. d, IL-5 and IL-13 levels measured in BAL fluid 20 days p.i. with C. neoformans (as in Fig. 5c); Data from 1 experiment, n = 4 mice per group. e, f, Frequency of activated lung CD4 + T cells making cytokines assessed on day 20 post C. neoformans infection of either full Il1r1-/- DOCK8 KO mice in 2 independent experiments (e), or in mice with lack of IL-1R expression restricted to T cells in 3 independent experiments (f); In (e) n = 8 WT, 10 KO, and 12 KO x Il1r1-/-; In (f) n = 9 KO, 13 Tcrb-/- recipients, and 12 Tcrb-/-Dock8-/- recipients. g, Dock8-/- mice were treated with Q-VD-OPh with or without administration of IL-1β during C. neoformans infection and number of activated CD4 T cells quantified in lung, as well as frequency of CD4 + T cells making cytokines 8 days after infection. Comparisons between all groups are non-significant unless indicated. For (c–f, h) error bars represent median ± min/max with box boundaries denoting 25th and 75th percentiles; for (g) means shown for each group; each data point is from an individual mouse. Statistical analyses were performed using: two-tailed Kruskal-Wallis ANOVA (c, eg) or Mann-Whitney test (d). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Extended Data Fig. 6 Cytokine response during GM-CSF blockade and expression of GM-CSF receptor.

a, Dock8-/- mice were treated with αGMCSF during infection with C. neoformans and on day 20 p.i., the frequency of CD4+ T cells producing cytokines evaluated in lung compared to WT and untreated KO mice. Data from 3 independent experiments; n = 12 WT, 18 KO, and 24 treated KO. Error bars represent group medians ± min/max with box boundaries denoting 25th and 75th percentiles. Statistical analyses were performed using two-tailed Kruskal-Wallis ANOVA. **P < 0.01 and ***P < 0.001. b, c, GM-CSF receptor alpha chain (CD116) expression levels on lung cell populations p.i. with C. neoformans on either day 20 (b), or on day 8 (c). Representative histograms shown of 3 independent experiments.

Extended Data Fig. 7 Working model of the mechanisms driving the type-2 CD4 + T cell bias in absence of Dock8.

In tissue, Dock8-deficient mononuclear phagocytes (MNPs) expressing CX3CR1 are highly sensitive to migration-induced shattering (cytothripsis). The shattering of these cells results in caspase activation, which leads to IL-1β release, a necessary but not sufficient signal in the Th2 bias. IL-1β acts directly on CD4 + T cells via IL-1R and induces GM-CSF production. GM-CSF likely acts back on MNPs, increasing pro-IL-1β expression. The type-2 response of CD4 + T cells is abolished by inhibiting IL-1R signaling on T cells or by blocking caspase activation, and is substantially reduced when GM-CSF is neutralized.

Supplementary information

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Supplementary Video 1

Four-dimensional dynamic image series (×100 magnification) of activated T cells from WT (left) and Dock8−/− (right) LifeAct-GFP+ mice migrating in 3.6 mg ml−1 collagen matrices, acquired with Zeiss ×10 air objective. Two-dimensional maximum intensity rendering is shown. Rectangular boxes indicate instances of KO cell stretching. Video represents 67 frames (1 frame min−1) shown at ~11 frames s−1. Total movie length is 67 min.

Supplementary Video 2

Four-dimensional dynamic image series (×100 magnification) of LPS-stimulated BMDCs from WT (left) and Dock8−/− (right) LifeAct-GFP+ mice migrating in 1.7 mg ml−1 collagen matrices, acquired with Zeiss ×10 air objective. Two-dimensional maximum intensity rendering is shown. Video represents 211 frames (1 frame min−1) shown at ~23 frames s−1. Total movie length is 211 min.

Supplementary Video 3

Four-dimensional dynamic image series (×200 magnification) of LPS-stimulated BMDCs from Dock8−/− LifeAct-GFP+ mice migrating in 1.7 mg ml−1 collagen matrices, acquired with Zeiss ×20 water immersion objective. Two-dimensional maximum intensity rendering is shown. A representative WT and then KO BMDC are shown sequentially. Video represents 85 frames for WT (total length is 3 h), followed by 60 frames for KO (total length is 2 h), both at 1 frame 2 min−1, shown at ~10 frames s−1.

Source data

Source Data Fig. 5

BMDCs were incubated in medium or added to 1.7 mg ml−1 collagen matrices. After 40 min, expression of cell death pathway components was assessed by immunoblot (uncropped blots shown). Two independent replicates are shown per group (corresponding to Fig. 5a).

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Schneider, C., Shen, C., Gopal, A.A. et al. Migration-induced cell shattering due to DOCK8 deficiency causes a type 2–biased helper T cell response. Nat Immunol 21, 1528–1539 (2020). https://doi.org/10.1038/s41590-020-0795-1

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