Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: CXCR6–CXCL16 axis promotes NT CD4 TRM establishment. (A) Box plot showing the expression of Cxcr6 mRNA among CD4 TRM of the NT and lungs. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (B and C) Box plot showing the expression of Cxcr6 mRNA among different cell clusters of lungs and NT. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by Wilcoxon rank sum test with Benjamini–Hochberg correction. (C) Volcano plot for differential expressed genes in the NT in comparison with the lungs of the Th17 cluster. The dotted lines indicate fold change and adjusted P value cutoffs. (D) Bar plot with individual data points for the expression of CXCR6 (each median fluorescence intensity [MFI] normalized to mean MFI from CD4 TEM iv + of NT) in CD4 TRM and iv + CD4 TEM from the lungs and NT of naïve mice and PR8 IAV-infected mice (30 dpi) as indicated. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (E and F) Expression of CXCR6 on OT-II CD4 TRM of NT and lung on day 30 following PR8-OVA infection as indicated. (E) Representative histograms for CXCR6 expression from OT-II CD4 TRM of NT and lung are shown. (F) Bar plot with individual data points showing the expression of CXCR6 (each MFI normalized to mean MFI of NT OT-II CD4 TRM). The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (G–K) Frequency of different cell populations within different organs derived from WT and Cxcr6 −/− BM chimeric mice on day 30 after infection with PR8 IAV as indicated. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots showing the percentage of WT and Cxcr6 −/− CD4 TRM in NT and lungs. (I) Bar plot with individual data points for the percentage of WT and Cxcr6 −/− CD4 TRM in NT and lungs as indicated. (J) Bar plot with individual data points for the percentage of I-A b NP 306–322 tetramer-specific WT and Cxcr6 −/− CD4 TRM in NT and lungs as indicated. Samples that had <7 OT-II CD4 TRM were excluded from the analysis. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (K) Bar plot with individual data points showing the percentage of Cxcr6 −/− and WT CD4 T cells in different organs of the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (L) A representative microscopic image of CXCL16 expression (magenta) and OT-II CD4 T cells (red and green) in the olfactory epithelium of the murine NT. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Scale bar: 100 μm for main image and 50 μm for the inset. (M–O) Antigen-specific CD4 TRM in the lungs and NT of mice treated with isotype control or anti-CXCL16 antibody on day 10 after PR8 IAV infection. (M) Schematic representation of the experimental setup. (N) Representative flow cytometry plots indicating the percentage of I-A b NP 306–322 tetramer-specific CD4 TRM are shown. (O) Bar plot with individual data points indicating the absolute number of I-A b NP 306–322 tetramer-specific CD4 TRM. The experiment was performed twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test.

Article Snippet: B6.SJL- Ptprc a Pepc b /BoyJ (CD45.1 + ) and B6.129P2- Cxcr6 tm1Litt /J ( Cxcr6 −/− ) mice were purchased from the Jackson Laboratory, USA, and were bred at EBM.

Techniques: Expressing, Two Tailed Test, Comparison, Fluorescence, Infection, Derivative Assay, Flow Cytometry, Olfactory, Isolation, Control

Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: Additional characterization of experimental models, related to Figs. 6, 7, and 8. (A) Percentage of CD45 + donor cells (both Cxcr6 −/− and WT) and recipient cells in the blood of BM chimera on day 67 following BM transplantation. Left panel: Bar plot with individual data points for the percentage of donor cells and recipient cells. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by one-way ANOVA, with Tukey’s multiple comparison test. Right panel: A representative flow cytometry plot showing the percentage of donor cells and recipient cells. (B) Bar plot with individual data points showing the percentage of donor and recipient cells among CD4 T cells in different organs in the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Representative microscopic image of CXCL16 expression (magenta) in the olfactory epithelium of the murine NT is shown. Two negative controls for CXCL16 staining showing NT stained (1) with α-rabbit secondary IgG Texas red only and (2) stained with isotype control and α-rabbit secondary IgG Texas red. Hoechst is indicated in grey. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Top images are stitched to show the whole NT. Scale bar: 500 μm for top panels and 100 μm for bottom. (D) Gating strategy to identify CD4 TEM (CD4 + CCR7 − CD45RA − ), CD4 TCM (CD4 + CCR7 + CD45RA − ), naïve CD4 Tc (CD4 + CCR7 + CD45RA + ), CD4 TEMRA(CD4 + CCR7 − CD45RA + ), and CD4 TRM (CD4 + CCR7 − CD45RA − CD69 + ) in NT and blood. (E) RORγt + CD4 + T cells in the PPs of WT C57BL/6 mice, CD4 cre Rorc fl/wt , and CD4 cre Rorc fl/fl mice. Left panel: Bar plot with individual data points showing the frequency of RORγt + CD4 + T cells among all CD3 + T cells. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the percentage of RORγt + CD4 + T cells in different groups. (F) Microscopy images (magnified) for TUNEL + cells in the nasal septum (respiratory region) from CD4 cre Rorc fl/wt and CD4 cre Rorc fl/fl mice (same as ). Scale bar: 50 μm. (G and H) Microscopy for TUNEL + cells and viral titer (TCID 50 /g) from organs of mice infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The mice were treated with isotype or IL-17a/f antibody. (G) Bar plot with individual data points showing number of TUNEL + cells in the nasal septum (respiratory region) on day 4 after X31 IAV infection. The experiment was repeated twice. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. (H) Viral titer (TCID 50 /g) from NT and lungs on day 3 after X31 IAV infection. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA.

Article Snippet: B6.SJL- Ptprc a Pepc b /BoyJ (CD45.1 + ) and B6.129P2- Cxcr6 tm1Litt /J ( Cxcr6 −/− ) mice were purchased from the Jackson Laboratory, USA, and were bred at EBM.

Techniques: Transplantation Assay, Comparison, Flow Cytometry, Infection, Expressing, Olfactory, Staining, Control, Isolation, Two Tailed Test, Microscopy, TUNEL Assay

Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: Functional IAV-specific CD4 TRM exist in the nasopharynx of healthy human subjects. (A) Bar plot with individual data points showing the frequency of CD4 Tc in the NT and PBMC. The experiment was performed seven times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by unpaired two-tailed t test. (B) Bar plot with individual data points showing the frequency of naïve CD4 Tc (CD4 + CCR7 + CD45RA + ), CD4 TCM (CD4 + CCR7 + CD45RA − ), CD4 TEM (CD4 + CCR7 − CD45RA − ), CD4 TEMRA (CD4 + CCR7 − CD45RA + ), and CD4 TRM (CD4 + CCR7 − CD45RA − CD69 + ) in NT and blood. NS, not significant; ****P < 0.0001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C and D) Expression of CD103 and CXCR6 on NT CD4 TRM and CD4 TEM of PBMC derived from healthy human subjects. (C) Representative flow cytometry plots indicating the percentage of CD103 and CXCR6 expression on CD4 TRM and CD4 TEM from the same subject. (D) Bar plot with individual data points showing the percentage of CD103 and CXCR6 expression on CD4 TRM and CD4 TEM. Each data point indicates one subject. The experiment was performed five times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by unpaired two-tailed t test. (E) Percentage of NT CD4 TRM and CD4 TEM from PBMC-expressing IL-17a, IFN-γ, and TNFα. The cytokine expression is indicated from unstimulated cells and IAV NP peptide pool and M1 peptide pool-stimulated cells connected by a line. Each line on the graph indicates cytokine expression from the cells of each subject. NS, not significant; ****P < 0.0001; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. (F) Bar plot with individual data points showing the percentage of NT CD4 TRM and CD4 TEM from PBMC-expressing IL-17a, IFN-γ, and TNF after stimulation with IAV NP peptide pool and M1 peptide pool (after subtraction of signals from the unstimulated control; negative values considered zero). The experiment was performed seven times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (G) Representative flow cytometry plots showing the expression of IL-17a in NT CD4 TRM and CD4 TEM of PBMC, which are stimulated with IAV NP peptide pool and M1 peptide pool or left unstimulated. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA.

Article Snippet: B6.SJL- Ptprc a Pepc b /BoyJ (CD45.1 + ) and B6.129P2- Cxcr6 tm1Litt /J ( Cxcr6 −/− ) mice were purchased from the Jackson Laboratory, USA, and were bred at EBM.

Techniques: Functional Assay, Two Tailed Test, Comparison, Expressing, Derivative Assay, Flow Cytometry, Control

Journal: Redox Biology

Article Title: Aryl hydrocarbon receptor in club cells drives Th17-mediated lung injury following inhalation exposure to environmentally persistent free radicals

doi: 10.1016/j.redox.2026.104105

Figure Lengend Snippet: Generation and validation of club cell-specific AHR knockout mice ( Ahr ΔCC). (a) Schematic of breeding strategy to generate Ahr ΔCC mice by crossing Ahr fl/fl mice with Scgb1a1-CreER TM mice, followed by tamoxifen induction. (b) Immunofluorescence staining showing club cell marker CC10 (green), AHR (red) and DAPI (blue) in lung sections of Cre-negative Ahr fl/fl LM control (top panel) and Ahr ΔCC (bottom panel) mice. (c) Representative flow cytometry plots of lung epithelial cells gated as CD45 − CD31 − EpCAM + CC10 + cells isolated from lungs of LM (left) or Ahr ΔCC (center) mice and Fluorescence-minus-one (FMO) control for AHR staining (right). (d) Quantification of the percentage of AHR + CC10 + cells in the lungs of LM (white bar) and Ahr ΔCC (gray bar) mice. Data represent mean ± SEM, n = 3-4 mice per group. Statistical significance was determined using Student's t-test; ∗p < 0.05.

Article Snippet: Male Ahr tm3.1Bra /J mice carrying a floxed exon 2 allele of the Ahr gene (JAX stock #006203) and female B6N.129S6(Cg)- Scgb1a1 tm1(cre/ERT)Blh /J mice expressing tamoxifen-inducible Cre recombinase under the control of the club cell-specific Scgb1a1 promoter (JAX stock #016225) were obtained from Jackson Laboratory.

Techniques: Biomarker Discovery, Knock-Out, Immunofluorescence, Staining, Marker, Control, Flow Cytometry, Isolation, Fluorescence

Journal: bioRxiv

Article Title: Endocytosis at the mouse blood brain barrier is elevated during sleep

doi: 10.64898/2026.04.02.716170

Figure Lengend Snippet: a. Imaging of injected Dextran Alexa Fluor 594 in BECs of mice with wild type Dynamin 2 expressed by AAV-BR1-DNM2-GFP. b. Imaging of injected Dextran Alexa Fluor 594 in BECs expressing dominant negative dynamin with AAV-BR1-DNM2_K44A-GFP. c. Fluorescence intensity of dextran 594 in the BECs of Dynamin 2and Dynamin 2_K44A expressing mice. n=11 and 13. d. Fluorescence intensity of dextran 594 in the blood of Dynamin 2 and Dynamin 2_K44A expressing mice. n=3 and 4. e. Dynamin 2_K44A dominant negative mutant in the BECs increases sleep in mice. n=5. f. Quantification of sleep time in wide type and Dynamin 2_K44A mice. n=5 mice per group. h. Imaging of injected Dextran Alexa Fluor 647 in BECs of wild type mice and Dynamin 2 knockout mice. i. Imaging of injected Dextran Alexa Fluor 647 in the BBB of mice with Dynamin 2 knocked out. j. Fluorescence intensity of dextran 647 in the blood of Dynamin II and Dynamin 2 KO mice. Fluorescence intensity of dextran in brain endothelial cells of DNM2 flox/flox and dynamin 2 knock out mice. n=5. k. Knock out of Dynamin 2 in the BECs increases sleep in mice. n=4 in each group. l. Quantification of sleep time in wild type and Dynamin 2 knock out mice. n=4 in each group. fl, flox. ZT, Zeitgeber Time. DNM, Dynamin. Scale, 5 μm. *** P <0.001; ** P <0.01; * P <0.05.

Article Snippet: The B6.129S1(Cg)- Dnm2 tm .1Pdc /J mice were obtained from Jackson laboratory (#013542, Jackson lab) and were born, bred and housed at University of Pennsylvania, Smilow Research Center of Translation animal facility.

Techniques: Imaging, Injection, Expressing, Dominant Negative Mutation, Fluorescence, Knock-Out

Journal: bioRxiv

Article Title: Endocytosis at the mouse blood brain barrier is elevated during sleep

doi: 10.64898/2026.04.02.716170

Figure Lengend Snippet: a. Immunostaining of DNM2 in BECs from control DNM2 flox/flox mice. b. Immunostaining of DNM2 in BECs from AAV-BI30-Cre-GFP mediated DNM2 knockout mice. c. Imaging of Exo70 in BECs of control mice. d. Imaging of Exo70 in BECs of Exo70 knock out mice. DNM2, Dynamin 2. Scale, 5 μm. *** P <0.001; ** P <0.01; * P <0.05

Article Snippet: The B6.129S1(Cg)- Dnm2 tm .1Pdc /J mice were obtained from Jackson laboratory (#013542, Jackson lab) and were born, bred and housed at University of Pennsylvania, Smilow Research Center of Translation animal facility.

Techniques: Immunostaining, Control, Knock-Out, Imaging