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An αvβ3 integrin checkpoint is critical for efficient TH2 cell cytokine polarization and potentiation of antigen-specific immunity

Abstract

Naive CD4+ T lymphocytes initially undergo antigen-specific activation to promote a broad-spectrum response before adopting bespoke cytokine expression profiles shaped by intercellular microenvironmental cues, resulting in pathogen-focused modular cytokine responses. Interleukin (IL)-4-induced Gata3 upregulation is important for the helper type 2 T cell (TH2 cell) polarization associated with anti-helminth immunity and misdirected allergic inflammation. Whether additional microenvironmental factors participate is unclear. Using whole mouse-genome CRISPR–Cas9 screens, we discovered a previously unappreciated role for αvβ3 integrin in TH2 cell differentiation. Low-level αvβ3 expression by naive CD4+ T cells contributed to pan-T cell activation by promoting T–T cell clustering and IL-2/CD25/STAT5 signaling. Subsequently, IL-4/Gata3-induced selective upregulation of αvβ3 licensed intercellular αvβ3–Thy1 interactions among TH2 cells, enhanced mammalian target of rapamycin (mTOR) signaling, supported differentiation and promoted IL-5/IL-13 production. In mice, αvβ3 was required for efficient, allergen-driven, antigen-specific lung TH2 cell responses. Thus, αvβ3-expressing TH2 cells form multicellular factories to propagate and amplify TH2 cell responses.

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Fig. 1: Optimization of a TH2 cell culture protocol compatible with CRISPR screening.
Fig. 2: TH2 cells differentially express αvβ3 integrin which is required for optimal responses in the lung.
Fig. 3: Naive CD4+ T cell expression of αvβ3 promotes initial T cell activation and proliferation.
Fig. 4: IL-4/Gata3-mediated αvβ3 upregulation is required for TH2 cell differentiation in vitro.
Fig. 5: Multiple transcriptomic and signaling perturbations in αv-deficient TH2 cells revealed by genome-wide transcriptomic analyses and chemical compound treatment.
Fig. 6: Thy1–αvβ3 interaction promotes TH2 cell differentiation in vitro.

Data availability

All high-throughput data in the present study were deposited at the Gene Expression Omnibus under accession no. GSE179210. TH cell transcriptomic data were obtained from Th-express (https://th-express.org). Source data are provided with this paper.

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Acknowledgements

We thank the ARES staff, genotyping facility, flow cytometry core and the National Institute for Health and Care Research, Cambridge Biomedical Research Centre Cell Phenotyping hub for their technical assistance. Funding: the present study was supported by grants from the UK Medical Research Council (MRC; grant no. U105178805) and Wellcome Trust (grant nos. 100963/Z/13/Z and 220223/Z/20/Z). A.C.H.S. was supported by the Croucher Foundation. M.D.K. was supported by an MRC CARP award (MR/T005386/1). We thank the National Institutes of Health Tetramer Core Facility for supplying the 2W1S-MHC-II tetramer and D. Withers for providing the Cd4CreERT2 mice.

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Contributions

A.C.H.S. designed and performed experiments and wrote the paper. A.C.F.F., J.M., P.A.C., M.W.D.H., A.C., M.S., H.E.J., P.K. and M.D.K. performed experiments, provided advice on experimental design and interpretation, and commented on the manuscript. A.N.J.M. supervised the project, designed the experiments and wrote the paper.

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Correspondence to Aydan C. H. Szeto or Andrew N. J. McKenzie.

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Extended data

Extended Data Fig. 1 Optimisation of a genome-wide screen for regulators of TH2 cell differentiation.

Extended Data Fig. 1 (a) Schematic of the optimised TH2 cell culture protocol for CRISPR screening. (b) Flow cytometric analysis of IL-13Tom expression by TH2 cells transduced with NT or Gata3 sgRNAs using the optimised protocol. Data representative of 3 independent experiments. (c) RNA-sequencing analysis of Gata3-targeted versus non-targeted TH2 cells using the optimised screening protocol. (d) KEGG pathway analysis of genes downregulated in Gata3-targeted versus non-targeted TH2 cells. (e) and (f) Gene set enrichment analyses of genome-wide positive regulators of TH2 cell differentiation identified in the screen. (g) Selection of the top 1018 genes from the genome-wide screen for a secondary screen (fold change > 0.06 and p-value < 0.07). (h) Validation of novel regulators by individual confirmatory sgRNA knockdown. Data representative of 3 independent experiments; mean ± SD; one-way ANOVA with Dunnett’s post-hoc test. (i) Validation of novel regulators as in (h) with corresponding TH1 comparisons. (h) & (i) **** P < 0.0001, ***P = 0.0006 (Fermt3), **P = 0.0018 (Hsp90b1), 0.0053 (Tfap4), *P = 0.0102 (Fnta), 0.0169 (Smarcc1), 0.0212 (Apbb1ip), 0.0252 (Kmt2d).

Extended Data Fig. 2 Differential integrin expression by in vitro and in vivo TH cells.

Extended Data Fig. 2(a) Flow cytometric analysis of αv and β3 expression by TH2 cells in vitro. (b) Flow cytometric analysis of α4 (Itga4) and β1 (Itgb1) expression by TH cells in vitro. Data are representative of 2 independent experiments with 4 biologically independent samples in each experiment; unpaired two-sided t-test; ****P < 0.0001, ***P = 0.0004. (c) Gating strategy for TH cell subsets in the papain-challenged lung. (d) Flow cytometric gating strategy and quantification of lymphoid populations in naive mice. (e) Flow cytometric analysis of αv and β3 expression in control, αv- or β3- deficient naïve CD4+ T cells. (f) and (g) Quantification of lymphoid populations in naive mice. Data representative of 2 independent experiments with 3 biologically independent samples in each experiment; mean ± SD; unpaired two-sided t-test. (h) Schematic of the experimental induction of type 2 inflammation in the mouse lung with OVA/Alum. (i) Schematic of the experimental induction of type 2 inflammation in the mouse lung with papain. (j) Schematic of the experimental induction of type 1 inflammation in the mouse lung with LPS.

Extended Data Fig. 3 Gating strategy of TH cell populations in vivo.

Extended Data Fig. 3(a) & (b) Flow cytometric gating strategy for cytokine- and transcription factor- expressing THeff cells in the mediastinal lymph node of OVA/Alum-challenged mice. (c) Flow cytometric gating strategy for 2W1S-tetramer-specific TH2 cells in the papain-challenged lung. (d) & (e) Flow cytometric gating and quantification of 2W1S-tetramer-specific IL-5 and IL-13 producing TH effector cells in PMA/ionomycin stimulated lung lymphocytes. Data are pooled from 2 independent experiments and represent mean ± SD (n = 6 mice in naïve and papain only control groups, n = 16 mice in Cd4Cre group, n = 15 mice in ItgavCD4KO group); unpaired two-sided t-test; *P = 0.0418 (top, IL-5) and 0.0211 (bottom, IL-13). (f) Flow cytometric gating strategy for IFN-γ expressing THeff cells in the mediastinal lymph node of LPS-challenged mice.

Extended Data Fig. 4 Genome-wide transcriptomic analysis of av-deficient TH cells.

Extended Data Fig. 4(a) Principal component analysis of the transcriptomes of control versus αv-deficient TH cells. (b) Volcano plot depiction of differentially expressed genes in control versus αv-deficient TH2 cells. (c) & (d) RNA expression of type 2 genes by control or αv-deficient TH cells; Il13 ***P = 0.0002, **P = 0.0012; Il5 ***P = 0.0006, **P = 0.0025; Il4 **P = 0.0046, *P = 0.0167, Gata3 ****P < 0.0001. (e) RNA expression of type-1 signalling genes by control or αv-deficient TH cells; Stat1 ****P < 0.0001, *P = 0.0438; Isg20 *P = 0.0148; Runx3 **P = 0.0052, *P = 0.0220. (c) - (e) mean ± SEM; one-way ANOVA with Tukey’s post-hoc test. (f) Flow cytometric analysis of Runx3 expression by αv-deficient TH cells. Data are representative of 2 independent experiments; unpaired two-sided t-test; ***P = 0.0003. (g) KEGG pathway analysis of genes downregulated in αv-deficient versus control TH2 cells.

Extended Data Fig. 5 avb3-Thy1 inhibition does not affect TH1 cell differentiation and IFN-g expression.

Extended Data Fig. 5(a) Flow cytometric analysis of cytokine expression by TH1 cells cultured in the presence of vehicle (DMSO) or cilengitide. Data are representative of 3 independent experiments with 5 biologically independent samples in each experiment; mean ± SD; unpaired two-sided t-test; ***P = 0.0001 (top, % IFN-γ + cells) and 0.0005 (bottom, IFN-γ MFI). (b) Flow cytometric analysis of cytokine expression by TH1 cells cultured in the presence of isotype or anti-αv antibody. Data are representative of 2 independent experiments with 5 biologically independent samples in each experiment; mean ± SD; unpaired two-sided t-test; ****P < 0.0001. (c) Flow cytometric analysis of cytokine expression by TH1 cells cultured in the presence of isotype or anti-β3 antibody. Data are representative of 2 independent experiments with 5 biologically independent samples in each experiment; mean ± SD; unpaired two-sided t-test. (d) Flow cytometric analysis of cytokine expression by TH1 cells cultured in the presence of isotype or anti-Thy1 antibody. Data are representative of 2 independent experiments; paired two-sided t-test. (e) Flow cytometric analysis of Thy1-Fc conjugation to epoxy beads. (f) Flow cytometric gating strategy of CD4 T-T doublets in the mediastinal lymph nodes of OVA/Alum challenged mice. (g) Flow cytometric quantification of CD4 T-T doublets as in (f); *P = 0.0132 (Cd4Cre vs ItgavCD4KO), 0.0248 (Cd4Cre vs Itgb3CD4KO). (h) Flow cytometric quantification of type-2 cytokine expressing-CD4 T-T doublets as in (f); IL-5 + CD4 doublet: **P = 0.0021, *P = 0.0129; IL-13 + CD4 doublet: **P = 0.0046, *P = 0.0148. (g)(h) data are pooled from 2 independent experiments and represent mean ± SD (n = 12 mice in Cd4Cre and ItgavCD4KO groups, n = 11 mice in Itgb3CD4KO group); one-way ANOVA with Dunnett’s post-hoc test.

Extended Data Fig. 6 Proposed model for αvβ3-mediated potentiation of TH2 cell responses.

Naïve CD4+ T cells express low levels of integrin αvβ3 which contribute to T cell activation and signalling via the IL-2/CD25/STAT5 axis. IL-4-mediated Gata3 induction upregulates αvβ3 during TH2 cell differentiation, permitting intercellular interactions among TH2 cells via αvβ3-Thy1 binding. Such interactions enhance mTOR signalling and support optimal TH2 responses in vivo.

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Supplementary Table 1

Analyses of genome-wide and secondary screens by Wilcoxon-based hit calling.

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Source Data Fig. 6

Unprocessed immunmoblot of Thy1 detection.

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Szeto, A.C.H., Ferreira, A.C.F., Mannion, J. et al. An αvβ3 integrin checkpoint is critical for efficient TH2 cell cytokine polarization and potentiation of antigen-specific immunity. Nat Immunol 24, 123–135 (2023). https://doi.org/10.1038/s41590-022-01378-w

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