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: IAV-specific CD4 TRM are formed at the URT and are dependent on presence of cognate antigen. (A) Schematic diagram showing the procedure of transfer of OVA-specific (OT-II) CD4 T cells, infection of mice with PR8-OVA, and subsequent harvesting of organs. (B and C) CD45.1 + or tdTomato + OT-II CD4 TRM (gated on live CD3 + CD4iv − CD4 tissue + CD44 + CD62L − CD69 + as shown in ) in different organs of CD45.2 + recipients on day 30 following PR8-OVA infection. (B) Representative flow cytometry plots indicating the percentages of CD45.1 + OT-II CD4 TRM. The data are representative of one experiment out of three independent experiments. (C) Bar graph indicating the absolute number of CD45.1 + or tdTomato + OT-II CD4 TRM. 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 two-way ANOVA, with Tukey’s multiple comparison test. (D) Representative microscopic images of OT-II + (red) CD4 + (green) T cells in different sections of the NT and NALT out of two independent experiments. The sections are derived from day 30 following PR8-OVA infection of mice that received OT-II CD4 T cells as described in . A stitched microscopic image showing different regions of the NT and NALT is shown at the top right panel. Scale bar: 1 mm for the main image, 100 μm for insets I and II, and 20 μm for inset III. OE, olfactory epithelium; S, nasal septum. (E and F) Frequency of CD103 + and CD11a + cells among OT-II CD4 TRM in NT and lungs (gated on live CD3 + CD4iv − CD4 tissue + CD44 + CD62L − CD69 + ), and among total CD4 effector memory T cells (CD3 + CD4 iv + CD44 + CD62L − ) in blood of CD45.2 + recipients on day 30 following IAV infection. (E) Representative flow cytometry plots indicating the percentages of CD103 + and CD11a + cells. The data are representative of one experiment out of two independent experiments. (F) Bar plot with individual data points indicating the percentages of CD103 + and CD11a + cells. Each data point indicates an individual mouse. The experiment was repeated twice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA, with Dunnett’s multiple comparison test. (G–I) Kinetics of I-A b NP 306–322 tetramer, I-A b HA 91–107 tetramer-specific CD4 TRM, and OT-II + CD4 TRM in the NT and lungs on indicated days following PR8 or PR8-OVA IAV infection. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots indicating the percentages of I-A b NP 306–322 tetramer and I-A b HA 91–107 tetramer-specific CD4 TRM are shown. (I) Bar plot with individual data points indicating the absolute numbers of I-A b NP 306–322 tetramer, I-A b HA 91–107 tetramer-specific CD4 TRM, and OT-II + CD4 TRM. Each data point indicates an individual mouse. The experiment was performed twice, and the results (mean + SEM) were plotted. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA on log(x + 1)-transformed counts, with Dunnett’s multiple comparison test. (J) Bar plot with individual data points showing the percentage of Ki-67 + cells among I-A b NP 306–322 tetramer + and I-A b HA 91–107 tetramer + CD4 TRM in lung and NT isolated on day 10, 30, and 60 after PR8 infection. The experiment was performed twice, and the results (mean ± SEM) were 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. (K–M) Frequency of OT-II CD4 TRM in the NT on day 22 following infection with PR8 or PR8-OVA. (K) Schematic representation of the experimental setup. (L) A representative flow cytometry plot indicating the frequency of OT-II CD4 TRM. (M) Bar plot with individual data points showing the frequency of OT-II 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: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Infection, Flow Cytometry, Comparison, Derivative Assay, Olfactory, Transformation Assay, Isolation, Two Tailed Test

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: Characterization of CD4 TRM in lungs and NT after IAV infection. (A) Gating strategy to identify OT-II + CD4 TRM in the NT. (B) Representative microscopic images of OT-II + (red) CD4 + (green) T cells in NALT and nasal septum. Hoechst is indicated in blue. The sections are derived from day 30 following PR8-OVA infection of mice that received OT-II CD4 T cells as described in . Scale bars are 50 μm for NALT and 20 μm for septum. Please note some nonspecific red and green staining of epithelial cell around the NALTs. (C and D) Expression of CD103 and CD11a (median fluorescence intensity) on OT-II CD4 TRM in NT and lungs and on total CD4 TEM of blood of CD45.2 + recipients on day 30 following IAV infection. 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 one-way ANOVA, with Tukey’s multiple comparison test. (D) Bar plot with individual data points indicating the percentage of I-A b NP 306–322 tetramer + , I-A b HA 91–107 tetramer + , and OT-II CD4 TRM in lung and NT on different dpi with PR8 OVA IAV i.n. Each data point indicates an individual mouse. The experiment was performed twice, and the results (mean + SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA on arcsine square-root transformed frequencies with Dunnett’s multiple comparison test. (E) Flow cytometry plot showing the percentage of OT-II + CD4 TRM in the NT and lungs from different days after PR8 OVA infection. (F) Flow cytometry plot showing the percentage of I-A b HA 91–107 PE tetramer + I-A b HA 91–107 BV421tetramer + CD4 TRM and I-A b human CLIP 87–101 PE tetramer + I-A b human CLIP 87–101 BV421 tetramer + CD4 TRM in the NT of mice on day 60 after PR8 infection. (G) Representative flow cytometry plots of Ki67 + cells among I-A b HA 91–107 tetramer + and I-A b NP 306–322 tetramer + CD4 TRM of lungs and NT isolated on day 10, 30, and 60 after PR8 infection. (H and I) Frequency of OT-II CD4 TRM in the lung on day 22 following infection with PR8 or PR8-OVA. (H) Bar plot with individual data points showing the frequency of OT-II 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. (I) A representative flow cytometry plot indicating the frequency of OT-II CD4 TRM.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Infection, Derivative Assay, Staining, Expressing, Fluorescence, Comparison, Transformation Assay, Flow Cytometry, Isolation, Two Tailed Test

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: NT CD4 TRM express cytokines upon antigen stimulation. (A and B) Expression of intracellular cytokines in CD4 TRM of NT and lungs upon restimulation with IAV NP peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice or from naïve mice. (A) Flow cytometry plots indicating the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF. (B) Bar plot with individual data points showing the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C and D) Frequency of IFNγ + cells among all lung and NT CD4 TRM isolated on day 30 after PR8 infection and that are stimulated with peptide pool (IAV NP versus SARS-CoV-2 N) pulsed splenocytes. (C) Representative flow cytometry plots showing the expression of IFNγ + cells among all lung and NT CD4 TRM. (D) Graph indicating the IFNγ + cells among CD4 TRM stimulated by IAV NP versus SARS-CoV-2 N pulsed splenocytes connected by a line. Each line indicates an individual mouse. The experiment was performed three times, and the results were pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. (E and F) Expression of ICOS and PD-1 on CD4 TRM on day 30 after PR8 IAV infection. (E) Representative flow cytometry plots of CD4 TRM-expressing ICOS and PD-1 in lungs and NT. (F) Bar plot with individual data points showing the percentage of CD4 TRM-expressing ICOS and PD-1. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Expressing, Isolation, Infection, Flow Cytometry, Comparison, Two Tailed Test

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: IAV-specific CD4 TRM of the NT are functional and exhibit immunodominance hierarchy similar to the lungs. (A) Flow cytometry plots indicating the percentage of CD4 TRM expressing IFN-γ, IL-2, and TNF with and without stimulation with IAV NP peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice. (B) Bar plot with individual data points indicating the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF after stimulation with IAV NP peptide pool, IAV HA peptide pool, IAV NA peptide pool, and IAV NS1 peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice. The experiment was performed thrice, and the results (mean ± SEM) were pooled. NS, not significant; ***P < 0.001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Bar plot with individual data points showing the expression of PD-1 and ICOS (MFI normalized to average MFI of respective markers on NT CD4 TRM) on CD4 TRM of lungs and NT on day 30 following infection with PR8 IAV. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; *P < 0.05 by unpaired two-tailed t test. MFI, median fluorescence intensity.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Functional Assay, Flow Cytometry, Expressing, Isolation, Infection, Comparison, Two Tailed Test, Fluorescence

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: NT CD4 TRM provide protection against heterosubtypic challenge. (A–C) I-A b NP 306–322 tetramer and I-A b HA 91–107 tetramer-specific CD4 TRM in the NT and lungs of mice that were infected with X31 i.n. or left uninfected on day 30 following PR8 infection. The mice were treated with FTY720, and the organs were analyzed 6 days after X31 infection. (A) Schematic representation of the experimental setup. (B) Bar plot with individual data points indicating the percentage of I-A b NP 306–322 tetramer + cells among CD4 TRM of lungs and NT. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (C) Representative flow cytometry plots indicating the percentages of I-A b NP 306–322 tetramer and I-A b HA 91–107 tetramer-specific CD4 TRM are shown. (D) Schematic representation of primary and secondary infection of mice, their treatment with anti-CD4 antibody, anti-CD8 antibody, or/and FTY720 on the indicated days, and collection of NT. (E) Left panel: Bar plot with individual data points showing the frequency of CD4 Tc and CD8 Tc among all live cells in the NT of mice treated with isotype control antibody or anti-CD4 and anti-CD8 antibody. The experiment was performed with four to five mice per group. The result is shown as (mean ± SEM). NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the frequency of CD4 and CD8 Tc among total live cells. (F) Scatter plot indicating viral titers (TCID 50 /g) in NT of mice on day 3 following secondary infection with X31 IAV. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by one-way ANOVA with Dunnett’s multiple comparison test. The outliers were identified and removed using Grubbs’ method (alpha = 0.05). One outlier each removed from all groups except for groups: PR8 + anti-CD8 + FTY720 and PR8 + anti-CD4. (G–J) Viral titer and frequency of I-A b NP 306–322 + CD4 TRM in the NT and lungs of mice who have undergone i.t. or i.n. PR8 infection followed by i.n. infection with X31 on day 30 after primary infection. The mice were treated with FTY720 +/− anti-CD4 antibody or left untreated. (G) Schematic representation of the experimental set up. (H) Bar plot with individual data points indicating the frequency of I-A b NP 306–322 tetramer + CD4iv − CD44 + CD62L − cells in the NT and lungs of mice on day 3 after secondary X31 infection +FTY720 treatment versus mice without secondary X31 infection and not treated with FTY720 as shown in . The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (I) Representative flow cytometry plots indicating percentages of I-A b NP 306–322 tetramer + CD4iv − CD44 + CD62L − cells in NT and lung of mice with and without secondary X31 infection (related to H). (J) Scatter plot showing viral titers (TCID 50 /g) in NT and lungs of mice on day 3 following secondary infection with X31 IAV who were infected with PR8 i.n. or intratracheally 30 days prior to X31 infection. The mice were treated with FTY720 i.p. as indicated in . The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by one-way ANOVA, with Dunnett’s multiple comparison test.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Infection, Two Tailed Test, Flow Cytometry, Control, Comparison

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: IAV-specific NT CD4 TRM are induced by vaccination and provide protection. (A) I-A b HA 91–107 tetramer-specific CD4 TRM in the NT and lungs of mice that were infected with X31 i.n. or left uninfected on day 30 following PR8 infection. The mice were treated with FTY720, and the organs were analyzed 6 days after X31 infection. Bar plot with individual data points indicating the percentage of I-A b HA 91–107 tetramer + cells among CD4 TRM in lungs and NT. The experiment was done twice, 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. (B) I-A b NP 306–322 tetramer-specific CD4 TRM in the NT and lungs of mice 6 days after X31 infection. The mice were treated with FTY720 from day −2 to day 5 after infection. Left: Bar plot with individual data points indicating the percentage of I-A b NP 306–322 tetramer + cells among CD4 TRM of lungs and NT. The experiment was done twice, and the results (mean ± SEM) were pooled. Right: Representative flow cytometry plots indicating the percentages of I-A b NP 306–322 tetramer-specific CD4 TRM are shown. (C) Percentage of CD4 + and CD8 + T cells in the blood of mice on day 3 following secondary X31 infection from different groups as indicated in the . The experiment was done twice and the results (mean ± SEM) were 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. (D) Bar plot with individual data points showing the frequency of I-A b NP 306–322 tetramer + cells among CD3 + T cells in the NT of mice treated with isotype control antibody or anti-CD4 and anti-CD8 antibody. The experiment was performed with four to five mice per group. The result is shown as mean ± SEM. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (E) Percentage of relative infection of MDCK cells by X31 IAV after incubation with serum derived on day 30 after infection of mice with PR8 or X31. The serum from mice immunized with irrelevant protein (COVID spike or streptavidin) was used as controls. The experiment was performed twice, and the results (mean ± SEM) are pooled. (F) Bar plot with individual data points indicating the absolute number of I-A b NP 306–322 + CD4 TRM in lungs and NT of mice on day 14 and day 30 after infection with PR8 intratracheally. The experiment was performed with five mice per group. The result is shown as mean ± SEM. 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. (G–I) I-A d M2e 2–17 tetramer + CD4 TRM in the NT of BALB/c on day 10 after third immunization with CTA1-DD or CTA1-3M2e-DD i.n. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots indicating the percentage of I-A d M2e 2–17 tetramer + CD4 TRM from different treatment groups. Staining with I-A d Human CLIP 87–101 tetramer (CLIP control) is used as the negative control. (I) Bar plot with individual data points indicating the percentage of I-A d M2e 2–17 tetramer + cells among all CD4 TRM. The experiment was repeated twice, 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. (J–M) I-A d M2e 2–17 tetramer + CD4 TRM in the NT and lungs of BALB/c on day 30 after last immunization with CTA1-3M2e-DD i.n. (J) Schematic representation of the experimental setup. (K) Bar plot with individual data points indicating the percentage of I-A d M2e 2–17 tetramer + cells among all CD4 TRM of lungs and NT. The experiment was repeated twice, 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) Representative flow cytometry plots indicating the percentage of I-A d M2e 2–17 tetramer + CD4 TRM and I-A d human CLIP 87–101 tetramer + CD4 TRM from lungs and NT. (M) Bar plot with individual data points indicating the percentage of I-A d M2e 2–17 tetramer + cells among all CD4 TEM of MLN and cLN in mice immunized as shown in . The experiment was repeated twice, 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. (N–P) Survival rate and weight loss of CTA1-3M2e-DD or CTA1-DD-immunized BALB/c mice that were infected with PR8 in URT-restricted manner and were treated with or without FTY720 and anti-CD4 antibody or anti-CD8 antibody or isotype control antibody. (N) Schematic representation of the experimental setup. (O) Kaplan–Meier survival curves of mice from different groups after PR8 infection. The experiment was repeated thrice, and the results were pooled. *P < 0.05 by log-rank Mantel–Cox test. Significant differences were found between CTA1DD versus CTA1M2eDD+Iso (*) and CTA1DD versus CTA1M2eDD+Iso+FTY720 (*). Comparisons between CTA1M2eDD+Iso versus CTA1M2eDD+a-CD4+FTY720/CTA1M2eDD+a-CD8+FTY720/CTA1M2eDD+a-CD4+a-CD8+FTY720 were run but nonsignificant. (P) Weight loss curve for mice from different treatment groups as indicated. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. Statistical comparisons between treatment groups (NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05) were performed using a linear mixed-effects model with comparisons of weight across treatment groups (averaged over time) using estimated marginal means and pairwise comparisons with Tukey’s honest significant difference adjustment. Statistical comparisons across groups for each day are reported in . (Q) Scatter plot indicating viral titers (TCID 50 /g) in NT and lungs of immunized mice as shown in on day 3 following infection with PR8 IAV. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. No significant difference was found between the groups by one-way ANOVA, with Dunnett’s multiple comparison test.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Infection, Two Tailed Test, Flow Cytometry, Comparison, Control, Incubation, Derivative Assay, Staining, Negative 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: scRNA-seq of CD4 TRM reveals differential cluster distribution between organs. (A) Schematic diagram showing the preparation of sorted antigen-specific cells from mice infected with a sublethal dose (25 μl) of PR8 IAV i.n. for generation of GEM using 10x chromium controller. (B) UMAP plot of unsupervised clustering for 15,301 CD4 TRM and other non-CD4 T cells isolated from lungs and NT of naïve and PR8 IAV-infected mice. (C) Dot plot representing mean expression of selected marker genes for each T cell cluster. Color intensity from blue to red indicates average expression of genes, and size of the dot depicts percentage of cells expressing the gene within the clusters. Only T cell clusters were included in the analysis. Selected gene signatures are as follows: Naïve CD4 T signature , Th1 signature , Tfh signature , Th17 signature , Treg signature , cytotoxic CD4 signature , IFN response signature , NKT cells signature ( ; ), Acutely activated signature ( ; ), Quiescent memory signature ( ; ), early activated signature ( ; ). (D) Density plot showing the expression of selected marker genes or gene signatures, projected on the same UMAP as in . Signatures were the same as defined in . (E) Bar graph showing proportion of each UMAP cluster divided by organ and antigen specificity. Only antigen-specific CD4 TRM from infected mice were included in the analysis.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Infection, Isolation, Expressing, Marker

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: scRNA-seq of CD4 TRM in the NT versus lungs. (A) Dot plot representing mean expression of top five marker genes for each T cell cluster, identified by the FindAllMarker function and ordered according to log2 fold change. Color intensity from blue to red indicates average expression of genes, and size of the dot depicts percentage of cells expressing the gene within the clusters. (B) Bar graph showing proportion of each UMAP cluster divided by organ. All cells from all mice were included in the analysis. (C) UMAP plot split by tissue (lung or NT) and infection status (IAV infected or naïve) and colored according to identified clusters. (D) UMAP plot split by tissue (lung or NT) and antigen specificity (I-A b NP 306–322 tetramer or I-A b HA 91–107 tetramer) and colored according to identified clusters. Grey dots indicate cells that were not specific for the selected antigen within the specific organ. Contour lines indicate the density of the cells. (E) Volcano plot showing differentially expressed genes upregulated in the NT versus lungs among different cell clusters. Areas shaded in red indicate genes upregulated in NT, while areas shaded in grey genes upregulated in lungs. The dotted lines indicate P value and log2 fold change cutoffs. A list of differentially expressed genes is reported in .

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Expressing, Marker, Infection

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: scTCR-seq of CD4 TRM reveals clonal sharing between organs. (A) UMAP plot split by tissue (lung or NT) and colored according to clone size for CD4 TRM in IAV-infected mice. Clones were binned into single clones (1 member; light blue), small (2 to 5 members; red), medium (6 to 19 members; green), and large (over 20 members; gold). Light grey dots represent cells where TCR clonotype could not be assigned. (B) UMAP plot split by tissue (lung or NT) and antigen specificity (I-A b NP 306–322 tetramer or I-A b HA 91–107 tetramer) and colored according to clone size for CD4 TRM in IAV-infected mice. Clones were binned into single clones (1 member; light blue), small (2 to 5 members; red), medium (6 to 19 members; green), and large (over 20 members; gold). Light grey dots represent cells where TCR clonotype could not be assigned. (C) Bar plot characterizing the top 34 most expanded clones. The y axis reports CDR3 sequences of TCRαβ, while the bar graphs show the number of cells divided according to cluster distribution, I-A b NP 306–322 tetramer + or I-A b HA 91–107 tetramer + , and organ specificity. n.d., not determined. (D) Tile plot indicating paired TRAV and TRBV genes usage split by tissue (lung or NT) and antigen specificity (I-A b NP 306–322 tetramer or I-A b HA 91–107 tetramer) in CD4 TRM of IAV- infected mice. The size of the tile is proportional to TRAV usage frequency. (E) Graph showing clonal relationship between lungs and NT from PR8 IAV-infected mice. Each circle indicates one clonal family, and size of the circle defines the size of the clonal family. Color of the circle denotes the antigenic specificity against I-A b NP 306–322 tetramer and against I-A b HA 91–107 tetramer. n.d., not determined. (F) Clonotype bias plot for clones with at least 6 cells. Clonotypes are colored according to predominant cluster. The red line highlights the null distribution (background distribution) for each clone size. The clones to the right of the red line are biased clones. (G) UMAP plot, split by tissue (lung or NT), depicting two biased clones. Cells belonging to the clonal family are colored according to the cluster, while other cells are in light grey. (H) UMAP plot, split by tissue (lung or NT), depicting the two most expanded clones. Cells belonging to the clonal family are colored according to the cluster, while other cells are in light grey.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Infection, Clone 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: 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: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

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: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

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: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Functional Assay, Two Tailed Test, Comparison, Expressing, Derivative Assay, Flow Cytometry, 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: NT harbors Th17 CD4 TRM that reduce local pathology. (A and B) Frequency of IL-17a + cells among CD4 TRM of NT and lungs upon restimulation with IAV immunodominant NP 306–322 peptide in comparison with unstimulated cells. The organs are isolated on day 30 following PR8 IAV infection of mice and from naïve mice. (A) A representative flow cytometry plot showing the percentage of IL-17a in lungs and NT. (B) Bar plot with individual data points indicating the frequency of IL-17a + cells among all CD4 TRM. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Percentage of IL-17a + cells among CD4 TRM of the lungs and NT that are stimulated with NP 306–322 peptide in the presence or absence of brefeldin A. Left panel: Graph showing expression of IL-17a from the cells that are stimulated in the presence or absence of brefeldin A connected by a line. Each line on the graph indicates the cells from individual mouse. The experiment was repeated twice, and the results are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. Right panel: Representative flow cytometry plots showing the expression of IL17-a among CD4 TRM of lungs and NT. (D and E) Viral titers in the NT of CD4 cre Rorct fl/fl and CD4 cre Rorc fl/wt mice on day 3 following secondary infection with X31 IAV. The mice were infected with PR8 and reinfected with X31 IAV on day 30 after primary infection and treated with fingolimod i.p. as indicated. (D) A schematic representation of the experimental setup. (E) Bar plot with individual data points showing viral titers (TCID 50 /g) in the NT of the two groups. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (F–H) Microscopy for TUNEL + cells in the nasal septum (respiratory region) derived from CD4 cre Rorct fl/fl and CD4 cre Rorc fl/wt mice. The mice were infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The tissues were isolated on day 4 and day 8 following X31 IAV infection. (F) A schematic representation of the experimental setup. (G) Representative microscopic images for TUNEL + cells (brown) in the nasal septum from mice on day 4 after X31 IAV infection are shown. Scale bar: 100 µm for the main image, 50 μm for insets. A magnified version of this image is shown in . (H) Bar plot with individual data points showing the number of TUNEL + cells per mm 2 in the respiratory region of the nasal septum. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (I) Microscopy for PR8 mCherry (yellow) in the nasal septum (respiratory region) of mice infected with PR8 mCherry virus. Scale bar: 50 μm for the main image, 10 μm for insets. The NT was isolated on day 3 after infection.

Article Snippet: For flow cytometry experiments with cells from blood, lungs, NALT, and NT, mice were injected with CD4 FITC or CD4 APC-Cy7 (RM4-4) antibody i.v. to label the circulating CD4 T cells.

Techniques: Comparison, Isolation, Infection, Flow Cytometry, Expressing, Two Tailed Test, Microscopy, TUNEL Assay, Derivative Assay, Virus

Journal: Cell reports

Article Title: Temporal dynamics of immune cell transcriptomics in brain metastasis progression influenced by gut microbiome dysbiosis

doi: 10.1016/j.celrep.2025.115356

Figure Lengend Snippet:

Article Snippet: Following viability stain, cells were blocked in the same manner as described for CITE-seq preparation, using FcR Blocking Reagent (Miltenyi Biotec, 130-092-575) and then stained with one or a combination of the following antibodies: rat anti-mouse CD3e-647 (BioLegend, 155609, KT3.1.1), hamster anti-mouse TCR γ/δ-PE (BioLegend, 118107, GL3), rat anti-mouse CD117(c-kit)-PE-Cy7 (BioLegend, 105813, 2B8), mouse anti-mouse CX3CR1–421 (BioLegend, 149023, SA011F11), rat anti-mouse CD192(CCR2)-510 (BioLegend, 150617, SA203G11), rat anti-mouse CD3-SparkBlue550 (BioLegend, 100259, 17A2), rat anti-mouse CD8-FITC (BioLegend, 100706, 53–6.7), rat anti-mouse CD4–711 (BioLegend, 100549, RM4–5), rat anti-mouse Ly-6C-785 (BioLegend, 128041, HK1.4), rat anti-mouse Ly-6G-650 (BioLegend, 127641, 1A8), rat anti-mouse CD186(CXCR6)-PerCP/Cyanine5.5 (BioLegend, 151120, SA051D1), rat anti-mouse CD11b-605 (BioLegend, 101237, M1/70), rat anti-mouse CD45-PacificBlue (BioLegend, 103125, 30-F11), rat anti-mouse CD45-PerCP/Cyanine5.5 (BioLegend, 103131, 30-F11), rat anti-CD3-FITC (BioLegend, 100203, 17A2), hamster anti-mouse TCR γ/δ-PE-Cy7 (BioLegend, 118123, GL3), rat anti-mouse CD279(PD-1)-421 (BioLegend, 135217, 29F.1A12), rat anti-CD25-PE (BioLegend, 102007, PC61), rat anti-CD4-APC-Cy7 (Tonbo, 25-0042-U025, RM4–5), rat anti-mouse CD8a-710 (Tonbo, 80-0081-U025, 53–6.7), rat anti-mouse CD3-PacificBlue (BioLegend, 100213, 17A2), rat anti-mouse CD4–570 (BioLegend, 100541, RM4–5), rat anti-mouse CD8a-APC-Cy7 (BioLegend, 100713, 53–6.7), hamster anti-mouse TCR γ/δ−711 (BioLegend, 118149, GL3), mouse anti-mouse NK-1.1-SparkRed718 (BioLegend, 156533, S17016D), rat anti-mouse CD25–750 (BioLegend, 102077, PC61), mouse anti-mouse CD159a(NKG2A B6 )-PE (BioLegend, 142803, 16A11), rat anti-mouse CD107a(LAMP-1)-PE-Cy7 (BioLegend, 121619, 1D4B), rat anti-mouse/human CD11b-650 (BioLegend, 101259, M1/70), rat anti-mouse CD8a-647 (BioLegend, 100727, 53–6.7), rat anti-mouse CD279(PD-1)-421 (BioLegend, 135221, 29F.1A12), rat anti-mouse FOXP3–647 (BioLegend, 126407, MF-14).

Techniques: In Vivo, Recombinant, Blocking Assay, Red Blood Cell Lysis, Multiplex Assay, Sequencing, Software, Amplification, Fluorescence

Journal: Cell reports

Article Title: Temporal dynamics of immune cell transcriptomics in brain metastasis progression influenced by gut microbiome dysbiosis

doi: 10.1016/j.celrep.2025.115356

Figure Lengend Snippet:

Article Snippet: Flow: rat anti-CD4-APC-Cy7 (RM4–5) , Tonbo , Cat#25–0042-U025; RRID: N/A.

Techniques: In Vivo, Recombinant, Blocking Assay, Red Blood Cell Lysis, Multiplex Assay, Sequencing, Software, Amplification, Fluorescence