Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: A On day 0, seven-weeks-old female BALB/c mice were either i.n. immunized with rAd-HA, rAd-NP, and rAd-IL-1β (each 2 × 10 8 particles) or infected with the H1N1 strain A/PR/8/34 (100 PFU). At the indicated time points, lymphocytes were isolated from lung tissues to evaluate T-cell phenotypes and functional responses. In addition, blood and BALF samples were collected at the specified times to assess systemic and local antibody responses. The localization and organization of different immune cells was examined by histological techniques. B At the indicated times, antigen-specific cells were identified by pentamer staining, and total numbers of HA 533-541 - and NP 147-155 -specific Pent + cells are depicted. The distribution of antigen-specific T cells within the iv - and iv + proportions are illustrated by different coloring. C Absolute numbers of circulating effector and memory T-cell subsets, and those of the T RM pool induced by mucosal vaccination or H1N1 infection are shown. Iv - T RM (KLRG1-) are colored in different shades of blue (regarding CD69 and CD103 expression), while effector T cells (T eff ; iv + KLRG1 + CD127 - ) are shown in black, and effector memory T cells (T EM ; iv + KLRG1 + CD127 + ), and central memory T cells (T CM ; iv + KLRG1- CD127 + ) are shown in gray. Each data set represents the mean + SEM of per group ( n = 6 mice per group/timepoint, except 150 dpi in the H1N1 group represents only n = 4). Statistical significances were analyzed by two-way ANOVA followed by Šídák’s multiple comparison test (*, p < 0.1; **, p < 0.01; ***, p < 0.001 (gray line: iv - )). ( A ) Created in BioRender. Tenbusch, M. (2025) https://BioRender.com/o92n191 .

Article Snippet: Cells of each sample were counted (Automated Cell Counter Luna, Logos Biosystems) and labeled with TotalSeq-C anti-mouse hashtag antibodies (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody (Cat: 155861), TotalSeq-C0302 anti-mouse Hashtag 2 Antibody (Cat: 155863), TotalSeq-C0303 anti-mouse Hashtag 3 Antibody (Cat: 155865), TotalSeq-C0304 anti-mouse Hashtag 4 Antibody (Cat: 155867), BioLegend; 0.5 μg for 2x10 6 cells) for multiplex single-cell sequencing analysis and stained with APC-labeled H-2K D NP 147-155 Pentamer (ProImmune, 1:40) for 30 min at 4 °C.

Techniques: Infection, Isolation, Functional Assay, Staining, Expressing, Comparison

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: A Lungs from rAd-immunized or H1N1-infected mice were obtained 18 days post-treatment, and HE-stained lung sections were scanned at 40x magnification. Airways were generally intact and solely H1N1 infection caused lymphocytic infiltration reflected by areas of lymphocyte accumulations, without neutrophils, monocytes, and macrophages (arrows). One representative example of four mice per group is shown. Scale bars in whole slide lung images are 1000 µm and scale bars in magnified images are 100 µm. B At the indicated time points, lungs of naïve, immunized, and infected mice were removed, embedded into O.C.T. compound, and finally stained with anti-CD4, anti-CD8, and anti-B220. Nuclei are shown in blue (Hoechst33342), CD4 + T cells are shown in yellow, CD8 + T cells are shown in green, and B cells are shown in red. C To identify the localization of antigen-specific CD8 + T cells in the lung parenchyma, lung sections from day 18 p.i. were primary stained with anti-CD8, followed by MHC-I pentamer staining. The signal intensity of bound APC-labeled pentamers was amplified with anti-APC-AF647, and anti-mouse IgG2b-AF647. Nuclei are shown in blue (Hoechst33342), CD8 + T cells are shown in green, and MHC-I pentamer-specific T cells are shown in red. Images were acquired on a Leica SP5X laser scanning confocal microscope using a 40x oil objective and are representatives from a collection of different slices of four mice per group ( n = 4). Scale bars in each image are 50 µm.

Article Snippet: Cells of each sample were counted (Automated Cell Counter Luna, Logos Biosystems) and labeled with TotalSeq-C anti-mouse hashtag antibodies (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody (Cat: 155861), TotalSeq-C0302 anti-mouse Hashtag 2 Antibody (Cat: 155863), TotalSeq-C0303 anti-mouse Hashtag 3 Antibody (Cat: 155865), TotalSeq-C0304 anti-mouse Hashtag 4 Antibody (Cat: 155867), BioLegend; 0.5 μg for 2x10 6 cells) for multiplex single-cell sequencing analysis and stained with APC-labeled H-2K D NP 147-155 Pentamer (ProImmune, 1:40) for 30 min at 4 °C.

Techniques: Infection, Staining, Labeling, Amplification, Microscopy

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: Previously, rAd-immunized or H1N1-infected mice were challenged with H3N2 (10,000 PFU) 56 days after the initial prime, and lymphocytes from lung tissues were isolated 44 days after the challenge (+ H3N2). One set of mice was not reinfected and served as the control group (- H3N2). Additionally, some lymphocytes were restimulated in vitro using MHC-II- or MHC-I-restricted peptides derived from HA and NP, and ICS was used for the functional identification. A , B Phenotypic differentiation between antigen-specific T eff , T EM , and T CM , and different subsets of T RM . The graphs show the total number of HA 533-541 - and NP 147-155 -specific Pent + CD8 + T cells. C Frequencies of cytokine-specific CD8 + T cells investigated in secondary infected mice (+ H3N2) compared to only primed mice (- H3N2) are shown. A – C Each data point represents an individual mouse, and bars represent the mean of the group ( n = 6 mice for rAd and H1N1 (-H3N2), 7 mice for H1N1 (+ H3N2) and 8 mice for rAd (+ H3N2). To compare statistical effects between unchallenged (- H3N2) and challenged (+ H3N2) mice of one group, statistical significances were analyzed by two-tailed Mann-Whitney test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: Cells of each sample were counted (Automated Cell Counter Luna, Logos Biosystems) and labeled with TotalSeq-C anti-mouse hashtag antibodies (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody (Cat: 155861), TotalSeq-C0302 anti-mouse Hashtag 2 Antibody (Cat: 155863), TotalSeq-C0303 anti-mouse Hashtag 3 Antibody (Cat: 155865), TotalSeq-C0304 anti-mouse Hashtag 4 Antibody (Cat: 155867), BioLegend; 0.5 μg for 2x10 6 cells) for multiplex single-cell sequencing analysis and stained with APC-labeled H-2K D NP 147-155 Pentamer (ProImmune, 1:40) for 30 min at 4 °C.

Techniques: Infection, Isolation, Control, In Vitro, Derivative Assay, Functional Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: A The survival of previously induced IAV-specific T RM was examined upon secondary inflammatory events by subsequent infection with the unrelated RSV (1 × 10 6 PFU) or by LPS treatment (10 µg) on day 56 after priming. Three weeks after the challenge, lymphocytes were isolated from lung tissues. One set of initially primed mice remained without secondary exposure (- RSV, - LPS, respectively) and served as the control group. B Body weight was measured for nine days following the RSV challenge, and values represent percentages of the initial weight on day 0. C Antigen-specific CD8 + T cells were identified by pentamer staining. Total numbers of HA 533-541 -, NP 147-155 -, and M2 82-90 -specific tissue-resident CD8 + T cells are shown. D Frequencies of non-circulating CD8 + T cells that are at least positive for the single expression of either CD107a, IFNγ, IL-2, or TNFα were assessed after in vitro restimulation. E Immunofluorescence staining of lung tissues from initially rAd-immunized or H1N1-infected mice reinfected with RSV on day 35 and collected an additional 35 days after challenge. Tissue sections were stained with anti-B220 (green), RSV M2 82-90 pentamer (yellow), and IAV NP 147-155 pentamer (red). Nuclei are shown in blue (Hoechst33342). Images were acquired on a Leica SP5X laser scanning confocal microscope using a 40x oil objective and are representatives from a collection of different slices of three mice per group ( n = 3). Scale bars in each image are 50 µm. C , D Each data point represents an individual mouse, and bars represent the mean of the group mean ± SEM per group ( n = 6 mice per group, only in (D) one sample in the group rAd (-RSV) was lost due to technical issues). Numbers above the columns show fold change between unchallenged (- RSV) and challenged (+ RSV) mice. Statistical significances were analyzed ( B) by two-way ANOVA followed by Tukey’s multiple comparison test, or ( C , D ) by one-way ANOVA followed by Tukey’s multiple comparison test (( B ): *, p < 0.05 H1N1 + RSV vs. naïve + RSV; ( C , D ); *, p < 0.1; ***, p < 0.001; ****, p < 0.001). ( A ) Created in BioRender. Tenbusch, M. (2025) https://BioRender.com/g83h450 .

Article Snippet: Cells of each sample were counted (Automated Cell Counter Luna, Logos Biosystems) and labeled with TotalSeq-C anti-mouse hashtag antibodies (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody (Cat: 155861), TotalSeq-C0302 anti-mouse Hashtag 2 Antibody (Cat: 155863), TotalSeq-C0303 anti-mouse Hashtag 3 Antibody (Cat: 155865), TotalSeq-C0304 anti-mouse Hashtag 4 Antibody (Cat: 155867), BioLegend; 0.5 μg for 2x10 6 cells) for multiplex single-cell sequencing analysis and stained with APC-labeled H-2K D NP 147-155 Pentamer (ProImmune, 1:40) for 30 min at 4 °C.

Techniques: Infection, Isolation, Control, Staining, Expressing, In Vitro, Immunofluorescence, Microscopy, Comparison

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: A On day 0, seven-weeks-old female BALB/c mice were either i.n. immunized with rAd-HA, rAd-NP, and rAd-IL-1β (each 2 × 10 8 particles) or infected with the H1N1 strain A/PR/8/34 (100 PFU). At the indicated time points, lymphocytes were isolated from lung tissues to evaluate T-cell phenotypes and functional responses. In addition, blood and BALF samples were collected at the specified times to assess systemic and local antibody responses. The localization and organization of different immune cells was examined by histological techniques. B At the indicated times, antigen-specific cells were identified by pentamer staining, and total numbers of HA 533-541 - and NP 147-155 -specific Pent + cells are depicted. The distribution of antigen-specific T cells within the iv - and iv + proportions are illustrated by different coloring. C Absolute numbers of circulating effector and memory T-cell subsets, and those of the T RM pool induced by mucosal vaccination or H1N1 infection are shown. Iv - T RM (KLRG1-) are colored in different shades of blue (regarding CD69 and CD103 expression), while effector T cells (T eff ; iv + KLRG1 + CD127 - ) are shown in black, and effector memory T cells (T EM ; iv + KLRG1 + CD127 + ), and central memory T cells (T CM ; iv + KLRG1- CD127 + ) are shown in gray. Each data set represents the mean + SEM of per group ( n = 6 mice per group/timepoint, except 150 dpi in the H1N1 group represents only n = 4). Statistical significances were analyzed by two-way ANOVA followed by Šídák’s multiple comparison test (*, p < 0.1; **, p < 0.01; ***, p < 0.001 (gray line: iv - )). ( A ) Created in BioRender. Tenbusch, M. (2025) https://BioRender.com/o92n191 .

Article Snippet: Similarly, APC-conjugated Pro5 MHC NP 147-155 Pentamer (ProImmune, 1:20) was pre-incubated with anti-APC-AF647 (clone 936809, R&D Systems, Cat: FAB8927R, 10 μg/ml).

Techniques: Infection, Isolation, Functional Assay, Staining, Expressing, Comparison

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: A Lungs from rAd-immunized or H1N1-infected mice were obtained 18 days post-treatment, and HE-stained lung sections were scanned at 40x magnification. Airways were generally intact and solely H1N1 infection caused lymphocytic infiltration reflected by areas of lymphocyte accumulations, without neutrophils, monocytes, and macrophages (arrows). One representative example of four mice per group is shown. Scale bars in whole slide lung images are 1000 µm and scale bars in magnified images are 100 µm. B At the indicated time points, lungs of naïve, immunized, and infected mice were removed, embedded into O.C.T. compound, and finally stained with anti-CD4, anti-CD8, and anti-B220. Nuclei are shown in blue (Hoechst33342), CD4 + T cells are shown in yellow, CD8 + T cells are shown in green, and B cells are shown in red. C To identify the localization of antigen-specific CD8 + T cells in the lung parenchyma, lung sections from day 18 p.i. were primary stained with anti-CD8, followed by MHC-I pentamer staining. The signal intensity of bound APC-labeled pentamers was amplified with anti-APC-AF647, and anti-mouse IgG2b-AF647. Nuclei are shown in blue (Hoechst33342), CD8 + T cells are shown in green, and MHC-I pentamer-specific T cells are shown in red. Images were acquired on a Leica SP5X laser scanning confocal microscope using a 40x oil objective and are representatives from a collection of different slices of four mice per group ( n = 4). Scale bars in each image are 50 µm.

Article Snippet: Similarly, APC-conjugated Pro5 MHC NP 147-155 Pentamer (ProImmune, 1:20) was pre-incubated with anti-APC-AF647 (clone 936809, R&D Systems, Cat: FAB8927R, 10 μg/ml).

Techniques: Infection, Staining, Labeling, Amplification, Microscopy

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: Previously, rAd-immunized or H1N1-infected mice were challenged with H3N2 (10,000 PFU) 56 days after the initial prime, and lymphocytes from lung tissues were isolated 44 days after the challenge (+ H3N2). One set of mice was not reinfected and served as the control group (- H3N2). Additionally, some lymphocytes were restimulated in vitro using MHC-II- or MHC-I-restricted peptides derived from HA and NP, and ICS was used for the functional identification. A , B Phenotypic differentiation between antigen-specific T eff , T EM , and T CM , and different subsets of T RM . The graphs show the total number of HA 533-541 - and NP 147-155 -specific Pent + CD8 + T cells. C Frequencies of cytokine-specific CD8 + T cells investigated in secondary infected mice (+ H3N2) compared to only primed mice (- H3N2) are shown. A – C Each data point represents an individual mouse, and bars represent the mean of the group ( n = 6 mice for rAd and H1N1 (-H3N2), 7 mice for H1N1 (+ H3N2) and 8 mice for rAd (+ H3N2). To compare statistical effects between unchallenged (- H3N2) and challenged (+ H3N2) mice of one group, statistical significances were analyzed by two-tailed Mann-Whitney test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: Similarly, APC-conjugated Pro5 MHC NP 147-155 Pentamer (ProImmune, 1:20) was pre-incubated with anti-APC-AF647 (clone 936809, R&D Systems, Cat: FAB8927R, 10 μg/ml).

Techniques: Infection, Isolation, Control, In Vitro, Derivative Assay, Functional Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice

doi: 10.1038/s41467-025-58931-y

Figure Lengend Snippet: A The survival of previously induced IAV-specific T RM was examined upon secondary inflammatory events by subsequent infection with the unrelated RSV (1 × 10 6 PFU) or by LPS treatment (10 µg) on day 56 after priming. Three weeks after the challenge, lymphocytes were isolated from lung tissues. One set of initially primed mice remained without secondary exposure (- RSV, - LPS, respectively) and served as the control group. B Body weight was measured for nine days following the RSV challenge, and values represent percentages of the initial weight on day 0. C Antigen-specific CD8 + T cells were identified by pentamer staining. Total numbers of HA 533-541 -, NP 147-155 -, and M2 82-90 -specific tissue-resident CD8 + T cells are shown. D Frequencies of non-circulating CD8 + T cells that are at least positive for the single expression of either CD107a, IFNγ, IL-2, or TNFα were assessed after in vitro restimulation. E Immunofluorescence staining of lung tissues from initially rAd-immunized or H1N1-infected mice reinfected with RSV on day 35 and collected an additional 35 days after challenge. Tissue sections were stained with anti-B220 (green), RSV M2 82-90 pentamer (yellow), and IAV NP 147-155 pentamer (red). Nuclei are shown in blue (Hoechst33342). Images were acquired on a Leica SP5X laser scanning confocal microscope using a 40x oil objective and are representatives from a collection of different slices of three mice per group ( n = 3). Scale bars in each image are 50 µm. C , D Each data point represents an individual mouse, and bars represent the mean of the group mean ± SEM per group ( n = 6 mice per group, only in (D) one sample in the group rAd (-RSV) was lost due to technical issues). Numbers above the columns show fold change between unchallenged (- RSV) and challenged (+ RSV) mice. Statistical significances were analyzed ( B) by two-way ANOVA followed by Tukey’s multiple comparison test, or ( C , D ) by one-way ANOVA followed by Tukey’s multiple comparison test (( B ): *, p < 0.05 H1N1 + RSV vs. naïve + RSV; ( C , D ); *, p < 0.1; ***, p < 0.001; ****, p < 0.001). ( A ) Created in BioRender. Tenbusch, M. (2025) https://BioRender.com/g83h450 .

Article Snippet: Similarly, APC-conjugated Pro5 MHC NP 147-155 Pentamer (ProImmune, 1:20) was pre-incubated with anti-APC-AF647 (clone 936809, R&D Systems, Cat: FAB8927R, 10 μg/ml).

Techniques: Infection, Isolation, Control, Staining, Expressing, In Vitro, Immunofluorescence, Microscopy, Comparison