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Allergic airway reactions rewired by PI3Kδ mutation

Lawrence P. Kane

J Exp Med (2026) 223 (4): e20252509.

https://doi.org/10.1084/jem.20252509

Published: 20 February 2026

View article titled, Allergic airway reactions rewired by PI3Kδ mutation

Journal Articles

Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling

Kathrynne A. Warrick, Anne Katrine Z. Johansen, Mengchi Jiao, Megan E. Linnemann, Irene Saha, Suh-Chin J. Lin, Charles N. Vallez, Thomas Hagan, Jeffery D. Molkentin, Chandrashekhar Pasare

J Exp Med (2026) 223 (4): e20251717.

https://doi.org/10.1084/jem.20251717

Published: 20 February 2026

View article titled, Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling

Includes: Supplementary data

Journal Articles

A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation

J Exp Med (2026) 223 (4): e20252154.

https://doi.org/10.1084/jem.20252154

Published: 20 February 2026

View article titled, A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4+ T cell signaling and differentiation

Includes: Supplementary data

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$Immune checkpoint inhibition drives myocarditis in a novel mouse model. (A) Schematic diagram illustrating the DTG tetracycline–repressor system, driven by a myosin heavy chain, α isoform (MHC-α) promoter, for inducible overexpression of OVA in the heart. (B) Representative western blot for OVA from hearts of WT, tetracycline-regulated transactivator (tTA), OVA TG, OVA DTG, and OVA DTG + doxycycline (DOX) mice. β-Tubulin included for loading control. (C) Experimental regimen of early DOX administration to inhibit OVA expression during embryonic and postnatal development in DTG animals (DOX was removed at 5 wk of age). Transfer of 105 purified naive OTI T cells (injected retro-orbitally) was followed by the administration of αCTLA-4 and αPD-1 mouse monoclonal antibodies (dual ICI, 200 μg IP each). (D) Representative flow cytometry contour plots of CD45.1 congenically marked TCR Vα2+ OTI cells in the heart at day 9 following the adoptive transfer of OTI cells for myocarditis induction (pre-gated on live CD45.2+CD90.2+CD8+ cells). (E) Quantification of OTI expansion indicated by cells per mg of cardiac tissue. (F) Quantification of cardiac-infiltrating immune cells measured via flow cytometric analysis of CD45.2+ cells (n = 3). (G) Representative histological images of hearts stained with H&E from DTG mice receiving adoptive transfer (no ICI) and DTG mice receiving αCTLA-4 and αPD-1 (ICI) following OTI transfer (+dual ICI). Scale bar, 200 μm. (H) Kaplan–Meier survival curve following transfer of 106 OTI cells, with or without dual ICI administration (n = 8–9). (I) Representative echocardiograms performed at day 7 after adoptive transfer. (J) Echocardiographic analysis of fractional shortening (%FS) (n = 8–9). “D-1” indicates baseline %FS prior to adoptive transfer. (K) Heart weight-to-body weight (HW/BW) ratio (n = 6–8). (L) Representative electrocardiograms recorded at day 7 after transfer. All data points in graphs represent biological replicates. Statistical analysis performed via one-way ANOVA (E and F) and Student’s t test (H, J, and K). P value determined using Mantel–Cox log-rank (H). All data shown are mean ± SEM (E, F, J, and K). Data are representative of two to three independent experiments. **P < 0.01, ***P < 0.001; ns, not significant. Source data are available for this figure: SourceData F1.$

Immune checkpoint inhibition drives myocarditis in a novel mouse model. (A)...

in Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling > Journal of Experimental Medicine

Published: 20 February 2026

Figure 1. Immune checkpoint inhibition drives myocarditis in a novel mouse model. (A) Schematic diagram illustrating the DTG tetracycline–repressor system, driven by a myosin heavy chain, α isoform (MHC-α) promoter, for inducible overexpression More about this image found in Immune checkpoint inhibition drives myocarditis in a novel mouse model. (A)...

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PD-1 blockade induces de novo priming and differentiation of antigen-specific CD8 T cells. (A and B) Representative flow cytometry contour plots and cell numbers showing the kinetics of OTI T cell expansion in the MedLN at day 4.5, 6, or 9 after adoptive transfer. (C and D) Representative flow cytometry contour plots and cell numbers showing the kinetics of OTI T cell expansion in the heart at day 4.5, 6, or 9 after adoptive transfer. Quantified in D. n = 3 for no ICI condition; n = 5 for ICI condition (B and D). Statistical analysis performed via Student’s t test for each time point. (E and F) Representative gating of CD44hi, granzyme B+ (GrzmB) endogenous CD8 T cells (Endo. CD8 T cell), and OTI T cells from MedLN and heart at day 6 or day 9 after adoptive transfer (E). Quantified in F. (G) Intracellular IFNγ staining of Phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated CD8 T cells isolated from MedLN or heart 5 h after stimulation in the presence of Brefeldin A. FMO indicates fluorescence minus one control. n = 4 DTG mice receiving OTI T cell transfer and ICI (both endogenous CD8 T cells and OTI T cells from each animal). (H) Representative flow cytometry plots indicating OTI (TCR Vα2+CD45.1+) T cell expansion in MedLNs at day 9 after adoptive transfer. Mice received three doses (200 μg) of αCTLA-4 antibody, αPD-1 antibody, both (ICI), or vehicle control on days 1–3. (I) Quantification of H (n = 3–5). Statistical analysis performed via one-way ANOVA. All data shown are mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001; ns, not significant.

PD-1 blockade induces de novo priming and differentiation ...

in Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling > Journal of Experimental Medicine

Published: 20 February 2026

Figure 2. PD-1 blockade induces de novo priming and differentiation of antigen-specific CD8 T cells. (A and B) Representative flow cytometry contour plots and cell numbers showing the kinetics of OTI T cell expansion in the MedLN at day 4.5, 6, More about this image found in PD-1 blockade induces de novo priming and differentiation ...

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Cardiac-specific CD8 T cells drive innate inflammation and myocardial damage. (A) Quantification of cardiac-infiltrating myeloid cells (CD45.2+CD11b+ and CD45.2+CD11c+) and total T cells (endogenous and OTI T cells, CD45.2+CD90.2+) determined by flow cytometry analysis of the total heart on day 9 after adoptive transfer. (B) Delineation of myeloid subsets including myeloid DCs (CD11b+CD11c+), lymphoid DCs (CD11b−CD11c+), neutrophils (Neut) (CD11b+CD11c−Ly6chiLy6Ghi), inflammatory monocytes (Inflamm. mono) (CD11b+CD11c−Ly6chiLy6Glo), and macrophages (Macs) (CD11b+F4/80+). Statistics included in Fig. S4 B. (C) Representative immunohistochemistry for myeloid cells (CD68-white) and cell outlines with wheat germ agglutinin (WGA) (green) with DAPI-stained nuclei (blue) from ICI and control hearts at day 9 after OTI T cell transfer. Scale bar, 100 μm. (D) qRT-PCR analysis of Il1b and Il6 mRNA expression relative to Gapdh expression from bulk cardiac tissue (n = 3–6). (D and E) qRT-PCR analysis of Nppa, Nppb, and Myh7 mRNA expression from bulk cardiac tissue (n = 3–6). Statistical analysis performed via one-way ANOVA (D and E). Depicted as mean ± SEM. Data are representative of two independent experiments (A–E). (F) Schematic of experimental outline for bulk RNA-seq analysis of FACS-purified immune cells (CD45.2+CD90.2−) isolated from hearts of ICI-treated or untreated mice on day 9 after adoptive transfer. (G) Heatmap of inflammatory genes in sorted cardiac-infiltrating myeloid cells from mice treated with ICI following OT-I–adoptive transfer, compared with isotype antibody–treated control mice. The heatmap displays row-wise Z-scores of DESeq2-normalized RNA-seq counts. Each column represents a biological replicate sample grouped by treatment, while genes are grouped by pathways. Data are representative of a single experiment with three biological replicates per condition (G). *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

Cardiac-specific CD8 T cells drive innate inflammation and myocardial damag...

in Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling > Journal of Experimental Medicine

Published: 20 February 2026

Figure 3. Cardiac-specific CD8 T cells drive innate inflammation and myocardial damage. (A) Quantification of cardiac-infiltrating myeloid cells (CD45.2⁺CD11b⁺ and CD45.2⁺CD11c⁺) and total T cells (endogenous and OTI T cells, CD45.2⁺CD90.2⁺) More about this image found in Cardiac-specific CD8 T cells drive innate inflammation and myocardial damag...

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CD8 T cell–derived TNF is required for ICI-induced myocarditis pathogenesis and mortality. (A) qRT-PCR analysis of Tnf mRNA expression from bulk cardiac tissue (n = 3–6). (B) Infiltration of leukocytes (represented as cell number per mg of tissue) quantified by flow cytometry at day 9 after adoptive transfer of either 105 TNF-sufficient (WT OTI) or 105 TNF-deficient (Tnf−/− OTI) T cells. (C) qRT-PCR analysis of Il1b mRNA expression from bulk cardiac tissue. (D) qRT-PCR analysis of Il6 mRNA expression from bulk cardiac tissue. n = 4 per condition. Statistical analysis performed via one-way ANOVA. Data depicted as mean ± SEM. (E) Experimental regimen for therapeutic targeting of TNF. Mice received standard three doses of ICI for myocarditis injection with additional administration of TNF neutralizing antibody (αTNF, 500 μg) on day 3 and day 6 following the adoptive transfer of 106 OTI cells. (F) Survival of DTG mice receiving Tnf+/+ (WT) OTI cells, with and without αTNF treatment, or Tnf−/− OTI cells following myocarditis induction with ICI. n = 4, WT OTI +ICI. n = 5, Tnf−/− OTI +ICI. n = 5, WT OTI +ICI +αTNF. (G) Representative electrocardiograms of mice following the development of fulminant myocarditis on day 7 after adoptive transfer. (H) Heart rate (beats per minute) averaged over a 2-min interval recorded at day 7 after transfer (n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

CD8 T cell – derived TNF is required for ICI-induced myocarditis pathogenes...

in Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling > Journal of Experimental Medicine

Published: 20 February 2026

Figure 4. CD8 T cell – derived TNF is required for ICI-induced myocarditis pathogenesis and mortality. (A) qRT-PCR analysis of Tnf mRNA expression from bulk cardiac tissue (n = 3–6). (B) Infiltration of leukocytes (represented as cell number More about this image found in CD8 T cell – derived TNF is required for ICI-induced myocarditis pathogenes...

Images

TNFR2 blockade protects against myocarditis and preserves antitumor efficacy of ICI. (A) Flow cytometry analysis of surface TNF staining in expanded OTI or endogenous CD8 T cell populations in the presence of ICI. Cells were purified from the MedLN at day 6 and stimulated for 5 h ex vivo with PMA/ionomycin. (B) MFI (mean fluorescence intensity) of membrane TNF staining in endogenous vs. OTI CD8 T cells. Data indicate independent T cell populations isolated from three mice. (C) qRT-PCR analysis of Tnfrsf1a (encoding TNFR1) mRNA expression from bulk cardiac tissue (n = 3–6). (D) qRT-PCR analysis of Tnfrsf1b (encoding TNFR2) mRNA expression from bulk cardiac tissue (n = 3–6). (E) Experimental regimen for therapeutic targeting of TNF with concurrent tumor burden and myocarditis. Following the establishment of MC38 tumors, mice received 106 OTI cells, a standard three doses of ICI for myocarditis induction, and the additional administration of αTNFR2-blocking antibody on day 9 and day 12 after tumor inoculation. (F) Survival of DTG mice with tumor inoculation and myocarditis induction, with and without αTNFR2 treatment (500 μg of blocking antibody per mouse per dose for two doses). n = 8–9 per condition. (G) Tumor volume was assessed in DTG mice receiving ICI therapy (administered on days 9, 10, and 11) in combination with either vehicle control or αTNFR2 treatment (administered on days 9 and 12). Additional cohort received no treatment vehicle control (n = 4–9). (H) Representative electrocardiograms of mice following the development of fulminant myocarditis on day 7 after adoptive transfer. Statistical analysis performed via Student’s t test (B), one-way ANOVA (C and D), and two-way ANOVA (G). P value determined using Mantel–Cox log-rank (F). All data shown are mean ± SEM. Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

TNFR2 blockade protects against myocarditis and preserves antitumor efficac...

in Immune checkpoint inhibitor–induced myocarditis is dependent on CD8 T cell–derived TNF and TNFR2 signaling > Journal of Experimental Medicine

Published: 20 February 2026

Figure 5. TNFR2 blockade protects against myocarditis and preserves antitumor efficacy of ICI. (A) Flow cytometry analysis of surface TNF staining in expanded OTI or endogenous CD8 T cell populations in the presence of ICI. Cells were purified More about this image found in TNFR2 blockade protects against myocarditis and preserves antitumor efficac...

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Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and Pik3cdE1020K/+ animals were sensitized intranasally with HDM extracts (200 μg on days 0 and 7; 50 μg on days 14, 16, and 18) and lungs examined on day 20. (A) Experimental outline. (B) H&E staining of paraffin-embedded lung sections. Top panel: Arrowheads indicate thickening of the alveolar septa; pound signs indicate iBALT. Bottom panel: Short arrows show lymphocytes, long arrows show macrophages, and yellow arrows show eosinophils. (C) CD4 T cell counts (liveCD45+TCRβ+CD4+CD8−) measured by flow cytometry. (D) Eosinophil numbers quantified from H&E-stained lung sections. (E) Neutrophil counts (liveLineage−CD11b+Ly6G+) from lungs of the indicated animals. (F) Airway resistance (Rrs) was measured with a flexiVent instrument as cmH2O.s/ml using the indicated concentrations of methacholine. Data in E are representative of two independent experiments with n = 3–4 for each group per experiment. (G) UMAP showing clusters of lung CD45+ immune cells analyzed by scRNAseq from naïve WT, naïve Pik3cdE1020K/+, HDM-treated WT, and HDM-treated Pik3cdE1020K/+ animals. Each cluster was assigned a cell type using SingleR and supervised analysis of gene expression specific to each cluster (Table S1). Cells from three mice were analyzed per genotype and condition. (H) Individual lung CD45+ immune cells identified by scRNAseq analyzed for enrichment of response to IFNγ gene set. Left: UMAP visualization of enrichment P values in lung CD45+ immune cells from the indicated mice; P values described in color scale below. Right: Frequencies of cells with indicated magnitudes of enrichment P values. Statistical comparison of all CD45+ cells from WT and Pik3cdE1020K/+ HDM-treated groups was performed (chi-squared test), comparing frequencies of cells with P < 0.0005. (I) HDM-specific IgG2a, IgG3, IgE, and IgG1 from the indicated mice. (J) CCL3, CCL5, and CXCL10 (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Data in C–E, I, and J are from n = 6–10 mice for each group, pooled from two independent experiments. Data in B are representative of two independent experiments. Unless otherwise indicated, statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and ...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 1. Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and Pik3cd^E1020K/+ animals were sensitized intranasally with HDM extracts (200 μg on days 0 and 7; 50 μg on days 14, 16, and 18) and lungs examined on day 20. (A) More about this image found in Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and ...

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Aberrant Th1 responses at the expense of Th2 immunity in Pik3cdE1020K/+lungs following HDM sensitization. (A) Scatter plot comparing gene expression in CD4 T cells from WT and Pik3cdE1020K/+ HDM-treated mice (cluster 1 from Fig. 1 G). DEGs upregulated in WT CD4 T cells in red, and genes upregulated in Pik3cdE1020K/+ CD4 T cells in blue. (B) CD4+ T cells from scRNAseq of total CD45+ lung immune cells (cluster 1 from Fig. 1 G) were re-clustered to identify five unique clusters (0–4) of CD4+ T cells. Left: UMAP showing distribution of clusters 0–4; identities of each cluster were assigned based on cluster-specific gene expression. Right: Proportion of cells from each cluster in indicated mice. (C) Seurat heatmap showing expression of cluster-defining genes for indicated populations. (A–C) Cells from three mice were analyzed per genotype and condition. (D) Representative flow cytometry plots of intracellular IFNγ and IL-5 expression in lung CD4+ T cells (liveCD45+TCRβ+CD4+CD8−) from indicated mice. (E–H) Frequencies of lung CD4+ T cells expressing (E) IL-5; (F) IL-4; (G) IFNγ; and (H) IL-2 from indicated groups. (D–H)n = 9–10 for each group, pooled from two independent experiments. (I–L) TCRα-deficient recipient mice were injected with 1 × 106 WT or Pik3cdE1020K/+ naïve CD4 T cells 14 days prior to HDM sensitization. HDM was administered intranasally as indicated. n = 9–10 for each group, pooled from two independent experiments. (I) Experimental design. (J) Cell counts of lung CD4 T cells (liveCD45+TCRβ+CD4+CD8−) from the indicated groups. (K) Frequencies of IFNγ+ (left) and IL-4/5/13+ (right) lung CD4 T cells from the indicated groups. Frequencies of IL-4/5/13+ cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45+CD3−NK1.1−CD19−CD11b+Ly6G−SiglecF+) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Aberrant Th1 responses at the expense of Th2 immunity in Pik3cd...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 2. Aberrant Th1 responses at the expense of Th2 immunity in Pik3cd ^E1020K/+ lungs following HDM sensitization. (A) Scatter plot comparing gene expression in CD4 T cells from WT and Pik3cd^E1020K/+ HDM-treated mice (cluster 1 from Fig. 1 More about this image found in Aberrant Th1 responses at the expense of Th2 immunity in Pik3cd...

Images

Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative flow cytometry plots showing IL-4 and IL-13 staining in Th2-polarized live CD4+ cells. Right: Percentages of IL-4+ and IL-13+ cells from the indicated mice. n = 15 for each group, from 15 independent experiments. (B) Left: Representative flow cytometry histograms showing GATA3 expression in Th2-polarized live CD4+ cells from the indicated mice. Right: Fold GATA3 expression (MFI normalized to WT) in Th2-polarized live CD4+ cells from the indicated mice. n = 14 for each group, from 14 independent experiments. (C) Left: Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4+ cells. Right: Percentages of IFNγ+ cells. (D) Left: Representative flow cytometry histograms showing Tbet expression in Th2 and WT control Th1-polarized live CD4+ cells. Right: Fold Tbet expression (MFI normalized to WT) in Th2-polarized live CD4+ cells from the indicated mice. (C and D)n = 14 for each group, from 14 independent experiments. (E) Percentages of IFNγ+ (left) and IL-13+ (right) cells over a time course of Th2 differentiation (0, 24, 48, and 72 h). n = 10 for each group, from 10 independent experiments. (F) Bulk RNAseq of WT and Pik3cdE1020K/+ naïve CD4 T cells and in vitro polarized Th2 cells. n = 3 biological replicates for each group. Volcano plots showing DEGs in red (WT upregulated) and blue (Pik3cdE1020K/+ upregulated); DEGs defined using fold change >1.5, P < 0.05. (G) Enrichment of hallmark pathways among DEGs comparing WT and Pik3cdE1020K/+ Th2 cells. (H) Time course of fold induction of pAKT(T308), pS6(S240/44), and pAKT(S473) in WT and Pik3cdE1020K/+ live CD4+ cells during Th2 differentiation (0, 24, 48, and 72 h), measured by flow cytometry. Fold induction calculated using MFIs of the indicated readouts normalized to the 0 time point of the corresponding genotype. n = 5–9 for each group, from five to nine independent experiments. Statistical comparisons were made using ratio paired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T c...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 3. Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative More about this image found in Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T c...

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Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cdE1020K/+CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages of IL-2+ Th2 cells. n = 9 for each group, from nine independent experiments. (B, C, and E) Time course analysis (0, 24, 48, 72 h) of IL-2 production, and pSTAT5(Y694) and CD25 during Th2 polarization, measured by flow cytometry. n = 7–10 for each group, from 7–10 independent experiments. (B) Percentages of IL-2+ cells over time from the indicated mice. (C) Fold induction of pSTAT5(Y694) over time from the indicated mice. Fold induction was calculated using pSTAT5(Y694) MFIs normalized to the 0 time point of the corresponding genotype. (D) Schematic describing experiments shown in F and G. (E) Fold induction of CD25 expression from the indicated mice. Fold induction was calculated using CD25 MFIs normalized to the 0 time point of the corresponding genotype. (F and G) Th2-polarized CD4 T cells from WT and Pik3cdE1020K/+ were rested in serum-free media for 4 h and subsequently stimulated with hIL-2 over the indicated time course (0, 15, 60, 120 min). n = 4–5 for each group, from four to five independent experiments. (F) Fold induction of pSTAT5(Y694) over time from the indicated mice, measured by flow cytometry. (G) Fold induction of pS6(S240/44) over time from the indicated mice, measured by flow cytometry. Fold induction was calculated using MFIs (pSTAT5(Y694) or pS6(S240/44)) normalized to the 0 time point of the corresponding genotype. (H–J) Naïve CD4 T cells were Th2-polarized in the presence or absence of αIL-2 blocking antibody (20 μg/ml). (H) Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4+ cells. (I) Percentages of IL-4+ (left), IL-13+ (middle), and IFNγ+ (right) cells from the indicated mice, in the presence or absence of αIL-2. n = 11 for each group, from 11 independent experiments. (J) Left: Representative flow cytometry plots showing pAKT(T308) in Th2-polarized live CD4+ cells in the presence or absence of αIL-2. Right: Fold induction of pAKT(T308) in Th2-polarized live CD4+ cells from the indicated groups. Fold induction was calculated by normalizing pAKT(T308) MFIs to WT control cells. n = 5 for each group, from five independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 4. Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd ^E1020K/+ CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages More about this image found in Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd...

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Inactivation of Foxo1 in Pik3cdE1020K/+CD4+T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr (Xie et al., 2021): significantly enriched gene sets colored in blue. Genes upregulated in HDM-treated Pik3cdE1020K/+ CD4 T cells relative to WT counterparts (Fig. 2 A) were used as input for pathway enrichment. (B) Time course (0, 24, 48, 72 h) of pFoxo1(S256) in Th2-polarized live CD4+ cells from indicated mice. Left: Representative flow cytometry plots. Right: Fold induction of pFoxo1(S256) over time. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to the 0 time point of the corresponding genotype. n = 8 for each group, from eight independent experiments. (C) GSEA comparing Th2-polarized WT and Pik3cdE1020K/+ transcriptomes for the expression of Foxo1-activated (left) and Foxo1-repressed (right) gene sets. (D) Gene expression heatmap (row z-score) showing normalized RPKM values of leading edge genes from Foxo1-activated and Foxo1-repressed GSEA described in Fig. 4 C. (E) Naïve CD4+ T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody. Left: Representative flow cytometry plots showing pFoxo1(S256). Right: Fold induction of pFoxo1(S256). Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 10 for each group, from 10 independent experiments. (F) GSEA comparing control and αIL-2–treated Th2-polarized CD4 T cell transcriptomes for the expression of Foxo1-activated (top) and Foxo1-repressed (bottom) gene sets in the indicated groups. (G and H) Naïve CD4 T cells were nucleofected with gRNA-Cas9 complexes containing NC or Foxo1-targeting gRNAs and differentiated under Th2 conditions. n = 9 for each group, from nine independent experiments. (G) Left: Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ T cells. Right: Percentages of IFNγ+ and IL-4+ cells in Th2-polarized cells. (H) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in live CD4+ T cells. Right: Percentages of IL-2+ cells in Th2-polarized cells from the indicated groups. (I and J) TCRα-deficient recipient mice were injected with 1 × 106 NC or Foxo1 gRNA-Cas9–nucleofected naïve CD4 T cells 14 days prior to HDM sensitization. n = 6–9 for each group, pooled from two independent experiments. (I) Experimental design. (J) Frequencies of IFNγ+ (left) and IL-2+ (right) lung CD4 T cells. Statistical comparisons used ratio paired t tests (B and E–G) or unpaired t tests (I). **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Inactivation of Foxo1 in Pik3cd ^E1020K/+...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 5. Inactivation of Foxo1 in Pik3cd ^E1020K/+ CD4 ⁺ T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr ( Xie et al., 2021 ): significantly More about this image found in Inactivation of Foxo1 in Pik3cd ^E1020K/+...

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Pik3cdE1020Kreshapes the epigenetic landscape of CD4+T cells. (A and B) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq (n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cdE1020K/+-specific, and common peaks. (B) WT and Pik3cdE1020K/+-specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cdE1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cdE1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cdE1020K/+) and non-DEGs from bulk RNAseq data (Fig. 3 C) were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cdE1020K/+). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cdE1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cdE1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cdE1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cdE1020K/+) and DEGs showing repressed CTCF peaks in Pik3cdE1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; Xie et al., 2021) of TFTs (ChEA; Lachmann et al., 2010) was performed using these two categories of DEGs. Adjusted P values (−log10) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cdE1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments (n = 3), quantified in Fig. S4 C. (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cdE1020K/+ cells. Data are representative of three independent experiments (n = 3), Fig. S4 D. NC, negative control. Source data are available for this figure: SourceData F6.

Pik3cd ^E1020K reshapes the e...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 6. Pik3cd ^E1020K reshapes the epigenetic landscape of CD4 ⁺ T cells. (A and B) Naïve CD4 T cells from WT and Pik3cd^E1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq (n = 3). A total of 71,040 peaks were More about this image found in Pik3cd ^E1020K reshapes the e...

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Fas-FasL signaling potentiates T cell activation, exacerbating CD4+T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4+ T cells, measured by flow cytometry (gated on live CD4+). n = 8 for each group, from eight independent experiments. (B) Fold surface FasL expression (MFI normalized to NC) on NC and Foxo1 gRNA-targeted naïve CD4 T cells cultured under Th2-polarizing conditions. n = 4 for each group, from four independent experiments. (C) Left: Th2 polarized cells (live CD4+) from WT and Pik3cdE1020K/+ animals were gated as FasLlow, FasLmid, and FasLhigh. Right: Linear regressions comparing FasL MFIs in each gate with percentages of IFNγ+ cells from the indicated groups. Correlation R2 and P values are indicated. n = 7 for each group, from seven independent experiments. (D–F) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas- or Fasl-targeting gRNAs and polarized under Th2 conditions. n = 8–10 for each group, from 8 to 10 independent experiments. (D) Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ T cells. (E) Percentages of IFNγ+ (left), IL-4+ (middle), and IL-13+ (right) in Th2-polarized live CD4+ T cells. (F) Fold induction of pAKT(T308) (left), pFoxo1(S256) (middle), and pS6(S240/44) (right) in Th2-polarized live CD4 T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. (G and H) NC, Fas, and Fasl gRNA-treated naïve CD4 T cells underwent Th2 polarization in the presence or absence of recombinant multimeric FasL (FasL-LZ), and phosphorylation of AKT(T308), Foxo1(S256), and S6(S240/44) was analyzed by flow cytometry. n = 6 for each group, from six independent experiments. (G) Representative flow cytometry histograms showing pS6(S240/44) staining in Th2-polarized live CD4+ T cells. Dashed lines represent cells cultured without FasL-LZ; solid lines show cells cultured with FasL-LZ. (H) Fold induction of pAKT(T308), pFoxo1(S256), and pS6(S240/44) in Th2-polarized (–/+ FasL-LZ) live CD4+ T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Fas-FasL signaling potentiates T cell activation, exacerbating CD4 ⁺...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 7. Fas-FasL signaling potentiates T cell activation, exacerbating CD4 ⁺ T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4⁺ T cells, measured by More about this image found in Fas-FasL signaling potentiates T cell activation, exacerbating CD4 ⁺...

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Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4+ cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4+ T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ+ Th2-polarized cells. (C) Th2-polarized cells were gated as FasLlow, FasLmid, and FasLhigh. Frequencies of IFNγ+ cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr (Xie et al., 2021), with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 8. Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cd^E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions in More about this image found in Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4...

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Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 (green) and αFas-AF555 (red), and colocalization (yellow) was measured. Data are representative of three individual experiments, n = 19–24 unique images for each group. Left: Representative images of CD3ε and Fas costaining from the indicated groups. Right: Quantification of % colocalization between CD3ε and Fas in the indicated groups. (B and C) FLIM-FRET microscopy of CD3ε-Fas interactions in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 alone (donor control) or costained with αCD3ε-AF488 (donor) and αFas-AF555 (acceptor), and FL was measured. Results are representative of three independent experiments. (B) Representative images from the indicated groups. Scale bars in images indicate 4 μm. (C) Left: FL (ns) measurements of individual pixels from ROIs on cells from indicated groups. Right: FRET efficiency (%) within ROI from individual cells from the indicated groups. n = 18 for each group. (D) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas-targeting gRNAs and stimulated with αCD3/CD28 in the presence of hIL-2 for 72 h. Cells were rested in serum-free media, treated with αCD3 (1 μg/ml) (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses for the indicated groups. (E) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, treated with αCD3 (1 μg/ml) in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time for the indicated groups. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses. Statistical comparisons were made using ratio paired t tests (D and E) and unpaired t tests (A and C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; NC, negative control; ROIs, regions of interest.

Fas interacts with the TCR complex and costimulates TCR signaling. (A) Con...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 9. Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with More about this image found in Fas interacts with the TCR complex and costimulates TCR signaling. (A) Con...

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PI3Kδ regulates CD4+T cell differentiation through integration of TCR, IL-2 receptor, and Fas signaling, driving Foxo1 inactivation and transcriptional reprogramming.

PI3Kδ regulates CD4 ⁺ T cell differentiation through integrati...

in A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4⁺ T cell signaling and differentiation > Journal of Experimental Medicine

Published: 20 February 2026

Figure 10. PI3Kδ regulates CD4 ⁺ T cell differentiation through integration of TCR, IL-2 receptor, and Fas signaling, driving Foxo1 inactivation and transcriptional reprogramming. More about this image found in PI3Kδ regulates CD4 ⁺ T cell differentiation through integrati...

Journal Articles

Next-generation CRISPR screens enable causal systems immunology

Hao Shi, Hongbo Chi

J Exp Med (2026) 223 (3): e20241266.

https://doi.org/10.1084/jem.20241266

Published: 19 February 2026

View article titled, Next-generation CRISPR screens enable causal systems immunology

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Biological applications of CRISPR perturbations and expanded modalities of bulk CRISPR screens in T cells. (A) Biological applications of CRISPR perturbations include the discovery of master regulator genes, identification of downstream mechanisms through genetic interaction screens, mapping of causal biological circuits via single-cell perturbation screens, and investigation of non–cell-autonomous effects through spatial screening approaches, which collectively help facilitate therapeutic engineering. (B) Bulk CRISPR-based screening approaches, including CRISPR KO, CRISPRi, and CRISPRa screens, have been widely used in T cells to probe key functional readouts, such as activation (e.g., CD69 and CD25 expression), proliferation, mTORC1 signaling, cytokine production, and Foxp3 expression. Emerging screening strategies, including ORF overexpression, targeted knock-in, base editing, prime editing, and RNA editing, are adding new layers of functional interrogation to these platforms. sKO, single knockout; NTC, nontargeting control; dgRNA, catalytically dead guide RNA; pegRNA, prime editing guide RNA; HA-GD2-28ζ CAR, GD2-targeting CAR with HA tag and CD28-CD3ζ signaling domain.

Biological applications of CRISPR perturbations and expanded modalities of ...

in Next-generation CRISPR screens enable causal systems immunology > Journal of Experimental Medicine

Published: 19 February 2026

Figure 1. Biological applications of CRISPR perturbations and expanded modalities of bulk CRISPR screens in T cells. (A) Biological applications of CRISPR perturbations include the discovery of master regulator genes, identification of More about this image found in Biological applications of CRISPR perturbations and expanded modalities of ...

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