Journal: Cancer Research

Article Title: GFER Represents a Target for Dual Disruption of Redox Homeostasis and Reactivation of the Immune Response in Pancreatic Adenocarcinoma

doi: 10.1158/0008-5472.CAN-24-4211

Figure Lengend Snippet: Immunogenicity increases upon GFER depletion in PDAC cells. A–E, Gene set enrichment analysis of RNA-seq data reveals the upregulation of the IFNα response ( A ), IFNγ response ( B ), TNFα signaling via NFκB ( C ), inflammatory response ( D ), and IL2–STAT5 signaling ( E ) pathways in GFER-depleted PATC 124 cells infected with sgGFER. A total of 100,000 PATC cells were cultured in six-well plates for 7 days and then measured. F, RNA-seq analysis demonstrates significant changes in gene expression in orthotopic tumor samples derived from 10,000 KPC (p53 mut) cells infected with nontargeting shRNA ( n = 2) as well as shGFER (shGFER#4, n = 2; shGFER#5, n = 3). Tumors were harvested when all tumors were of comparable size. Immune system–related genes are highlighted in red boxes. G, Protein levels of chemokines ( CXCL9, CXCL10 , and CXCL11 ) related to CD8 + T-cell infiltration in orthotopic shCTRL and shGFER KPC (p53 mut) tumors. Statistical analysis was performed using a two-tailed Student unpaired t test. Data represent the mean ± SD; n = 4 mice. H and I, Immunophenotyping of orthotopic shCTRL and shGFER KPC tumors. A total of 10,000 KPC cells were injected into the pancreas, and tumors were harvested for multicolor flow cytometry analysis. The percentage of CD8 + T cells, CD4 + T cells, NK cells (NK1.1+ cells), B cells (B220+ cells), macrophages (F4/80+ cells), and dendritic cells (DC; CD11c + cells) were identified in CD45 + cells ( H ). The percentage of PD-1–positive and granzyme B–positive cells were identified in CD8 + and CD8 + PD-1 + cells, respectively ( I ). Fibroblasts were identified as CD45 − EpCAM − αSMA + cells ( J ). Statistical analysis was performed using a two-tailed Student unpaired t test. Data represent the mean ± SD; n = 5 mice. K, Kaplan–Meier survival analysis of C57BL/6 mice orthotopically implanted with 10,000 KPC (p53 mut) cells infected with shCTRL or shGFER. Mice received either anti-CD8 antibody (10 mg/kg) or rat IgG2a starting 1 day prior to surgery and then intraperitoneally every 7 days for four cycles. Animals were euthanized when tumor volume reached ∼2000 mm 3 . Survival curves were analyzed by one-way ANOVA ( n = 10 per group). L, Growth curves of 10,000 KPC (p53 mut) cells subcutaneously implanted into C57BL/6 mice. Prior to implantation, KPC cells were infected with shCTRL or shGFER. Tumor-bearing mice were treated with IgG control or anti–PD-1 antibodies. Tumor volume was measured every 4 days for 36 days. Statistical analysis was performed using one-way ANOVA. Data represent the mean ± SD; n = 4–7 per group. M, Pancreatic cancer patient with anti–PD-1/anti-CD40 treatment prior to chemotherapy were selected from the PRINCE trial and divided in to GFER-high and -low groups. The overall survival analysis was done with log-rank test ( N = 17).

Article Snippet: We used 8-week-old male or female nude mice (Strain #002019, The Jackson Laboratory) and immunocompetent C57BL/6J mice (Strain #000664, The Jackson Laboratory) and CD8 knockout (KO) mice (Strain #002665, The Jackson Laboratory) to establish subcutaneous and orthotopic models, with n = 3–10 mice per treatment group.

Techniques: Immunopeptidomics, RNA Sequencing, Infection, Cell Culture, Gene Expression, Derivative Assay, shRNA, Two Tailed Test, Injection, Flow Cytometry, Control

Journal:

Article Title: Origins and functional basis of regulatory CD11c + CD8 + T cells

doi: 10.1002/eji.200839057

Figure Lengend Snippet: CD11c+CD8+ T cells exist in naive mice. (A) Purified CD8+ T cells from the spleens of young female B6 mice (n=12) were used for flow cytometry and co-expression of CD11c, 33D1, and DC-SIGN is shown. Numbers in each panel show percent positive cells. (B) Absolute CD11c+CD8+ T cell numbers isolated from various tissues (Spl, spleen; Ln, lymph nodes; Thy, thymus; Liv, liver; Bm, bone marrow) from young female B6 mice (n=12) were calculated from pre-counted viable cells and are shown as bar graphs (mean ± SD). (C) CD11c+CD8+ and CD11c-CD8+ T cells were purified from the spleens of young female B6 mice (n=12) ((AU: OK?)), labeled with CFSE, and cultured (1 × 105/well) in 48-well culture plates pre-coated with anti-CD3 (10 μg/ml) and soluble anti-4-1BB (5 μg/ml). After 3 days, cultures were washed and flow cytometry performed. (D) Purified CD8+ T cells from the spleens of young female B6 mice (n=6) were used for flow cytometry. Gates were set for either CD11c+CD8+ or CD11c-CD8+ cells; surface (or intracellular for Foxp3) expression of the indicated molecules was calculated and presented as histograms (mean ± SD). All data (A-D) are representative of at least three experiments.

Article Snippet: The unbound cell fraction was collected and further enriched using paramagnetic CD8 beads using the manufacturer's guidelines (Dynal Mouse CD8 kit, InVitrogen, Carlsbad, CA).

Techniques: Purification, Flow Cytometry, Expressing, Isolation, Labeling, Cell Culture

Journal:

Article Title: Origins and functional basis of regulatory CD11c + CD8 + T cells

doi: 10.1002/eji.200839057

Figure Lengend Snippet: Precursor analysis of CD11c+CD8+ T cells. (A) CD8+ T cell subsets with or without surface CD11c from naive OT-I mice (n=6) were purified as described in the Materials and methods section. (B) Purified CD11c-CD8+ and CD11c+CD8+ T cell subsets (1 × 105/well) from naive OT-I mice (n=6) ((AU: OK?)) were cultured in 48-well culture plates. Cellular activation was started by adding mitomycin C-treated (50 μg/ml; 20 min at 37°C) T-cell free SIINFEKL-pulsed (3 μM) spleen cells and anti-4-1BB (5 μg/ml). Cells were analyzed 6 days later by two-color flow cytometry. The experiments were performed at least three times. Shown are representative results obtained after gating on Vα2+ cells. (C) CD8+ T cell subsets, purified as in (A) from naive OT-I mice (n=4), were adoptively transferred (5 × 106) into syngeneic 4-1BB-/- mice. Activation was achieved by injecting SIINFEKL (0.5 mg) and anti-4-1BB (100 μg/day × 2 days). Six days later, spleen cells were collected and three-color flow cytometry performed gating on Vα2+ cells. One of three independent experiments is shown. (D,E) Purified CD11c-CD8+ T cell cultures from naive OT-I mice (n=6) were set up in 24-well culture plates, coated, and subjected to flow cytometry 3 days later. Where shown, cultures were supplemented with various agonists diluted (log2) as given. The highest concentration of the various agonists (represented as darkest shade) were as follows: anti-4-1BB (10 μg/ml), anti-CD28 (10 μg/ml), PMA (10 ng/ml), ionomycin (0.5 ng/ml), IL-2 (20 ng/ml), IL-7 (20 ng/ml), and IL-15 (200 ng/ml). Anti-CD3 was used at 10 μg/ml. Pooled data from three independent experiments are shown (mean ± SD). (F) CD8+ T cells were purified from naive OT-I mice (n=3) and adoptively transferred into syngeneic 4-1BB-/- mice. Recipient mice were treated i.v. additionally with a single dose of either SIINFEKL (0.5 mg) together with control Ig (cIg), anti-4-1BB (100 μg/day × 2 days) or both. On day 7, spleen cells were subjected to two-color flow cytometry. One of eight independent experiments is shown. (G) Absolute CD3+CD4+ T cell numbers from experiment (F) were calculated from pre-counted viable cells and are shown as histograms (mean ± SD). (H) CD11c+CD8+ and CD11c-CD8+ T cells were purified from the SIINFEKL/anti-4-1BB group described in (F) and then cell surface (CD25, CD103, CD122 and Fas) or intracellular (Foxp3, perforin, and granzyme B) expression determined by flow cytometry. Shown here are data pooled from 3 independent experiments containing at least 3 mice per group (mean ± SD).

Article Snippet: The unbound cell fraction was collected and further enriched using paramagnetic CD8 beads using the manufacturer's guidelines (Dynal Mouse CD8 kit, InVitrogen, Carlsbad, CA).

Techniques: Purification, Cell Culture, Activation Assay, Flow Cytometry, Concentration Assay, Expressing

Journal:

Article Title: Origins and functional basis of regulatory CD11c + CD8 + T cells

doi: 10.1002/eji.200839057

Figure Lengend Snippet: Purified CD4+CD25-, CD11c-CD8+, and CD11c+CD8+ T cells were obtained from spleens and peripheral lymph nodes of naïve B6 mice (n = 3). (A) Aliquots of purified CD8+ T cell subsets were subjected to flow cytometry to assess cell purity. Numbers in each panel represent percent positive cells. (B) Graded amounts of purified CD11c+CD8+ or CD11c-CD8+ T cells were co-cultured with CFSE-labeled CD4+CD25- (5 × 103) stimulated with anti-CD3/anti-4-1BB for 3 days. At the end of culture, cells were washed and flow cytometry performed. A total of 2000 events for each sample were analyzed for analysis. The extent of CD4+ T cell division without co-culture is showed by the broken line and that which occurred due to co-culture is shown in the bars. The data shown are pooled from three independent experiments containing 3 mice per group (mean ± SD). (C-E) BALB/c mice were treated i.v. with anti-CD3 (500 ng), anti-4-1BB (100 μg/day × 2 days) or both. Seven days later, (D) CD11c+CD8+ and (E) CD11c-CD8+ T cells were purified from spleens and co-cultured with purified naive CFSE-labeled CD4+CD25- T cells (1 × 105) at the indicated effector : responder ratios for 3 days. At the end of the experiment, cells were washed, labeled with anti-CD4-PE, and flow cytometry was performed. Shown is the cell division among gated CD4+ T cell populations cultured in the presence or absence of added CD8+ T cell subsets. The value 58.0, in each panel, is the degree of CD4+ T cell division without coculture and the other variable number is the extent achieved when CD8+ T cell subsets were added to cultures. Dotted histograms indicate CD4+ T cell division obtained without coculture and unbroken histograms are those that obtained with coculture. (A, D, E) One of three similar experiments is shown.

Article Snippet: The unbound cell fraction was collected and further enriched using paramagnetic CD8 beads using the manufacturer's guidelines (Dynal Mouse CD8 kit, InVitrogen, Carlsbad, CA).

Techniques: Purification, Flow Cytometry, Cell Culture, Labeling, Co-Culture Assay

Journal:

Article Title: Origins and functional basis of regulatory CD11c + CD8 + T cells

doi: 10.1002/eji.200839057

Figure Lengend Snippet: (A) Colitis was induced in 4-1BB-/- mice with TNBS. The mice were then further treated i.p. with anti-4-1BB (100 μg/day for 4 days and once on day 7) after colitis induction. Purified CD11c-CD8+ and CD11c+CD8+ T cells (5 × 106 of each CD8+ T cell subtype; purified populations are shown in the representative flow cytometry plots) were adoptively transferred into syngeneic 4-1BB-/- mice. (B) Mortality in the treated mice (n=3 per experimental group) was followed for 10 days. (C) Pooled splenocytes and lymph node cells were obtained from the experiment shown in B (n=3 per experimental group), flow cytometry was performed, and the results depicted as contour plots highlighting CD4+ and CD8+ T cell percentages. (B, C) One of two independent experiments is shown.

Article Snippet: The unbound cell fraction was collected and further enriched using paramagnetic CD8 beads using the manufacturer's guidelines (Dynal Mouse CD8 kit, InVitrogen, Carlsbad, CA).

Techniques: Purification, Flow Cytometry

Journal:

Article Title: Origins and functional basis of regulatory CD11c + CD8 + T cells

doi: 10.1002/eji.200839057

Figure Lengend Snippet: (A) 5 × 106 CD8+ T cells from OT-I mice were injected into the tail-veins of syngeneic 4-1BB-/- recipients (naïve). Mice were additionally treated i.v. with anti-4-1BB (100 μg/day × 2 days) and SIINFEKL (0.5 mg) (treated). On day 7, spleen cells were collected, CD11c+ DCs were isolated as mentioned in Materials and methods section. CD8+ T cell contamination was prevented by removal of CD8+ cells with microbeads to yield CD11c+CD8- DCs. Expression of IDO was analyzed by RT-PCR. One of three independent experiments is shown, with 3 mice per group per experiment. (B) Mice were treated as in A. Untreated 4-1BB-/- mouse spleen cells were used as controls (None). To inhibit IDO activity, slow-release 1-MT or control placebo pellets were placed under the skin (back of the neck) of the mice before cell transfer and treatment. CHOP expression in CD4-gated cell populations was performed by flow cytometry. One of two independent experiments is shown, with 3 mice per group per experiment. (C) Treatments with SIINFEKL, anti-4-1BB, placebo, and 1-MT were performed as in A and B. Seven days after treatment, spleen cells were collected and flow cytometry performed. One of three independent experiments is shown, with 3 mice per group per experiment. (D) Wild-type (Wt) and appropriate gene knockout mice were treated as in A. Seven days after treatment, spleens were removed, single cell suspensions prepared, and flow cytometry performed. One of two independent experiments is shown, with 3 mice per group per experiment. Broken circles indicate total CD4+ T cell proportions (%) found in individual treatment groups.

Article Snippet: The unbound cell fraction was collected and further enriched using paramagnetic CD8 beads using the manufacturer's guidelines (Dynal Mouse CD8 kit, InVitrogen, Carlsbad, CA).

Techniques: Injection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Flow Cytometry, Gene Knockout

Journal:

Article Title: Origins and functional basis of regulatory CD11c + CD8 + T cells

doi: 10.1002/eji.200839057

Figure Lengend Snippet: (A) Splenic DCs were isolated and treated with 1-MT and anti-4-1BB as described in the Materials and methods section. The DCs were then pulsed with SIINFEKL, mixed with naive OT-I CD8+ T cells, and adoptively transferred into fresh syngeneic 4-1BB-/- recipients. Activation of adoptively transferred cells was achieved by anti-4-1BB. (B) Contour plots depict cellular composition of splenocytes from treated mice (n=3). Numbers in each panel represent percent positive cells. One of two experiments is shown.

Article Snippet: The unbound cell fraction was collected and further enriched using paramagnetic CD8 beads using the manufacturer's guidelines (Dynal Mouse CD8 kit, InVitrogen, Carlsbad, CA).

Techniques: Isolation, Activation Assay

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: CD8 + T‐cell deletion mitigated injury‐related neointimal hyperplasia in mice. (A)‐ Experimental design. Eightweekold male CD8a +/+ and CD8a −/− ‐mice were randomly subjected to a sham operation (Sham) or a carotid artery ligation+cuff (L + C) operation, followed by 14 days of either nonstress (NonStress) or chronic stress (Stress) prior to tissue collection and analysis. (B) Representative immunostaining and quantitative data show the infiltrated CD8a + macrophages ( red ) with DAPI nuclear counterstain ( blue ). Scale bar: 200 μm. (D, E) Representative hematoxylin and eosin (H&E) and quantitative data show the neointima/media area ratio, neointimal area, and intimal cell number ( n = 6 per group). Data are mean ± SEM ( n = 6/group). The significance of differences among the groups was analyzed by a one‐way ANOVA (C, D). NS, not significant.

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Ligation, Immunostaining

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: CD8 + T‐cell deficiency attenuated the injured vascular collagen deposition, cell proliferation, and macrophage infiltration. CD8a +/+ and CD8a −/− mice were subjected to a sham or L + C operation respectively and were sampled 14 days later. (A) Representative Masson's trichrome and PCNA immunostaining images of collagen deposition and PCNA + cells in the sham and L + C injured carotid arteries of mice of both genotypes. Scale bar: 200 μm. (B) The results of the quantitative analysis of collagen deposition, including the collagen fiber area ( left ), relative collagen content ( middle ), and number of PCNA + cells in the injured arteries of the four experimental groups. (C, D) Representative immunofluorescence images and quantitative data for the numbers of infiltrated CD68 + macrophages ( red ) in the carotid arteries of the four experimental groups. Scale bar: 200 μm. (D) The quantification of CD68 + cell numbers per section. Data are mean ± SEM ( n = 6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B, D).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Immunostaining, Immunofluorescence

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: CD8 + T‐cell deficiency reduced the investigated molecular alterations in response to the L + C injury. (A, B) Representative western blot images and combined quantitative data for the levels of galectin3, AT1R, p‐Akt, pmTOR, and pP38MAPK in the carotid arteries of CD8a +/+ and CD8a −/− mice subjected to the sham or L + C double injury. (C) The quantitative polymerase chain reaction (qPCR) analysis revealed alterations in the levels of MCP1, ICAM1, VCAM1, MMP2, MMP9, cathepsin S, and cathepsin K in injured arteries of the mice in the four experimental groups. Data are mean ± SEM ( n = 6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B, C).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Western Blot, Real-time Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: Chronic stress enhanced injury‐reduced CD8 + T‐cell infiltration and neointimal formation in the CD8a +/+ mice. The CD8a +/+ mice underwent a sham operation, L + C injury alone, or stress plus the L + C injury for 2 weeks and were then subjected to sampling for the histological analysis. (A, B) Representative H&E staining images and quantitative data showing the neointima/media area ratio ( left ), neointimal area ( middle ), and intimal cell counts ( right ). (C, D) Representative immunofluorescence images and quantitative data provide the numbers of infiltrated CD8a cells ( red ) in carotid artery cross‐sections of three experimental groups. Scale bar: 200 μm. Data are mean ± SEM ( n = 6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B, D).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Sampling, Staining, Immunofluorescence

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: Stress accelerated IFN‐γ +/+ CD8 + T‐cell infiltration into the injured carotid artery tissues of CD8a +/+ mice. Double immunofluorescence used anti‐CD8a (red) and anti‐INF‐γ (green) antibodies was applied to identify the IFN‐γ +/+ CD8 + T cells in the three experimental groups. (A, B) Representative double immunofluorescence and quantitative data show the numbers of IFN‐γ +/+ CD8 + T cells in the uninjured and injured carotid artery tissues of the three experimental groups. Scale bar: 100 μm. Data are mean ± SEM ( n = 6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Immunofluorescence

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: CD8 + T‐cell deletion attenuated the stress/L + C–induced carotid artery neointimal hyperplasia. CD8a +/+ and CD8a −/− mice that had been subjected to chronic (14‐day) stress plus the L + C injury were subjected to sampling for the histological analyses. (A, B) Representative H&E, Masson's trichrome, and PCNA staining images and combined quantitative data show the neointima/media area ratio, neointimal area, intimal cell count, collagen fiber area, relative collagen content, and number of PCNA + cells in the intima of the two experimental groups. (C, D) Representative immunofluorescence images and quantitative data for the numbers of infiltrated CD68 ( red ) in carotid artery sections of the two experimental groups. Scale bar: 200 μm. Data are mean ± SEM ( n = 6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B, D).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Sampling, Staining, Cell Counting, Immunofluorescence

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: CD8 + T‐cell deficiency suppressed the investigated molecular alterations in the carotid arteries in response to the stress plus L + C injury. (A, B) Representative western blot images and combined quantitative data for the levels of galectin3, AT1R, pAkt, pmTOR, and pP38MAPK proteins in the carotid arteries of both experimental groups. (C, D) The qPCR analyses revealed the levels of MCP1, VCAM1, ICAM1, MMP2, MMP9, cathepsin K, and cathepsin S genes. Data are mean ± SEM ( n = 6/group). Significance was assessed by unpaired Student's t ‐test (B, C).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: IFN‐γ signaling promoted CD8 + T‐cell recruitment and neointimal formation in mice subjected to the 2‐week chronic stress conditions. IFN‐γ +/+ and IFN‐γ −/− mice that had received double injuries underwent the chronic stress and were then subjected to sampling for immunoblotting. (A, B) Representative western blot images and quantitative data show the levels of galectin‐3 and AT1R proteins in the carotid arteries of the mice in both experimental groups. IFN‐γ +/+ mice that had received double injuries underwent the stress+vehicle intervention or stress+IFN‐γ neutralizing antibody (Anti‐ IFN‐γ) intervention, respectively, for 2 weeks and were then sampled for the histological analysis. (C, D) Representative H&E staining and CD8a immunofluorescence images and quantitative data present the neointima/media area ratio, neointimal area, intimal cell counts, and CD8 + T cell numbers in the injured arteries of IFN‐γ +/+ mice. CD8a −/− mice that had received double injuries underwent stress plus an adoptive transfer with CD8 + T cells isolated from stressed IFN‐γ +/+ or stressed IFN‐γ −/− donor mice, respectively for 2 weeks and were then subjected to sampling for the histological analysis. (E, F) Representative H&E and CD8a immunofluorescence and quantitative data show the neointima/media area ratio, neointimal area, and intimal cell numbers in the carotid arteries of both experimental groups. Data are mean ± SEM ( n = 6/group). Significance was assessed by unpaired Student's t ‐test (B, D, F).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Sampling, Western Blot, Staining, Immunofluorescence, Adoptive Transfer Assay, Isolation

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: IFN‐γ depletion and deletion reduced CD8a + T‐cell infiltration into the injured carotid arteries of the stressed mice. (A, B) Representative immunofluorescence images and quantitative data for the numbers of infiltrated CD8a ( red ) in injured carotid artery sections of the vehicle and IFN‐γ depletion groups. (A, B) Representative immunofluorescence images and quantitative data for the numbers of infiltrated CD8a ( red ) in injured carotid artery sections of IFN‐γ +/+ CD8 + T‐cell and IFN‐γ −/− CD8 + T‐cell mice. Scale bar: 200 μm. Data are mean ± SEM ( n = 6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B, D).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Immunofluorescence

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: The effects of mouse S‐serum on VSMCs' intracellular signaling and their function. Mouse VSMCs were treated with 5% Non‐serum/CD8a +/+ , 5% Non‐serum/CD8a −/− , 5% S‐serum/CD8a +/+ , or 5% S‐serum/CD8a −/− for 12 h and then subjected to a western blotting assay. (A, B) Representative western blot images and combined quantitative data show the levels of p‐Akt and p‐mTOR in the four experimental groups ( n = 4 per group). Here, 5% Non‐serum/CD8a +/+ , 5% Non‐serum/CD8a −/− , 5% S‐serum/CD8a +/+ , and 5% S‐serum/CD8a −/− were respectively added to the out‐wells of transwells. Mouse VSMCs were seeded onto the inner chambers with serum‐free DMEM and then cultured (for 6 h for the migration assay or overnight for the invasion assay). (C, D) Representative microscopy and quantitative data show the numbers of migrated and invaded VSMCs. Scale bar: 75 μm. Data are mean ± SEM ( n = 4–6 for each group). Significance was assessed by a one‐way ANOVA followed by Tukey's post hoc tests (B, D).

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Western Blot, Cell Culture, Migration, Invasion Assay, Microscopy

Journal: The FASEB Journal

Article Title: CD8 + T‐Cell Deletion Suppressed the Development of Injury‐Induced Experimental Neointimal Hyperplasia in Mice With or Without Chronic Stress

doi: 10.1096/fj.202502380R

Figure Lengend Snippet: Graphical illustration of the mechanism of vascular remodeling under chronic stress. CD8+ T‐cell functions as an important mediator of injury‐related experimental neointimal hyperplasia via the modulation of inflammation, proteolysis and proliferation that is mediated by the IFN‐γ/AT1R‐galactin‐3 and mTOR/Akt‐p38MAPK axes. MCP‐1, monocyte chemoattractant protein‐1; ICAM‐1, intracellular adhesion molecular‐1; VCAM‐1, vascular cellular adhesion molecular‐1; MMP‐2, matrix metalloproteinase‐2, MMP‐9, matrix metalloproteinase‐9, Cat S, cathepsin S; Cat K, cathepsin K; AT1R, angiotensin type 1 receptor; p‐Akt, protein kinase‐B; p‐mTOR, phospho‐mammalian target of rapamycin; p‐p38, phospho‐p38 mitogen activation protein kinase.

Article Snippet: Male CD8a knockout mice (CD8a −/−19 ) and IFN‐γ knockout mice (IFN‐γ −/−19 ) (both on C57BL/6 background) were obtained from Shanghai Biomodel Organism Science & Technology Development Co. (Shanghai, China; protocol no. 2021‐W5‐2174).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Dietary environmental factors shape the defence against infection with Cryptosporidium

doi: 10.1101/2023.03.30.534739

Figure Lengend Snippet: (A) Representative image showing Ct- CR (yellow arrow-head pointing to mNeonGreen positive parasites on the luminal side of villi) and the close proximity of intraepithelial lymphocytes (magenta arrow pointing to CD3 + IELs) to infected epithelial cells in the ileum. (B) AHR-tdTomato expression by TCRαβ and TCRγδ CD8 + IEL subsets. (C&D) Ki67+ proliferating IELs in the Ct- CR infected WT mice. (E) Number of IELs in the small intestine of Ct- CR infected WT mice. (F) Intracellular IFNγ levels of TCRαβ and TCRγδ CD8 + IEL subsets. (G) Histogram of intracellular granzyme-B in TCRαβ and TCRγδ CD8 + IEL subsets. (A-G) Representative results of at least 2 independent experiments. Each dot represents individual mice. Error bars, mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as calculated by t-test or one-way ANOVA with Tukey post-test.

Article Snippet: Intermediate layer containing IELs was collected and washed with 1X PBS followed by either flowcytometry analysis or used for CD8α IELs purification using EasySepTM mouse CD8a positive selection kit-II (StemCell Technologies; Cat# 18953).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: Dietary environmental factors shape the defence against infection with Cryptosporidium

doi: 10.1101/2023.03.30.534739

Figure Lengend Snippet: (A) Representative flow cytometry plots showing the percentage of CD8α IEL subsets in WT and AHRKO Immune mice at steady state. (B & C) Percentage (B) and absolute number (C) of TCRαβ and TCRγδ CD8 + IEL subsets in AHRKO Immune mice and littermate WT controls at steady state. (D & E) Percentage (D) and absolute number (E) of CD8 + IEL subsets on day 7 post- Ct- CR infection (C). (D & E) Representative FACS plots (F) and percentage (G) of Ki67 positive CD8α IEL subsets in Ct- CR-infected mice. (H) Percentage of granzyme-B-expressing total CD8 + IELs (I) Mean fluorescence intensity (MFI) of intracellular granzyme-B in TCRαβ and TCRγδ CD8 + IEL subsets in AHRKO Immune mice and littermate WT controls infected with Ct- CR. (A-G) Representative results of at least 2 independent experiments. Each dot represents individual mice. Error bars, mean + SEM. ns-not significant, *p < 0.05, **p < 0.01, ***p < 0.001, as calculated by t-test.

Article Snippet: Intermediate layer containing IELs was collected and washed with 1X PBS followed by either flowcytometry analysis or used for CD8α IELs purification using EasySepTM mouse CD8a positive selection kit-II (StemCell Technologies; Cat# 18953).

Techniques: Flow Cytometry, Infection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Dietary environmental factors shape the defence against infection with Cryptosporidium

doi: 10.1101/2023.03.30.534739

Figure Lengend Snippet: (A) Experimental design of Rag2-IL2Rγ-CD47 triple knockout mice receiving CD8 + IELs followed by infection with Ct- CR. (B) Survival of Rag2-IL2Rγ-CD47 triple knockout mice infected with Ct- CR that received WT CD8 + IELs compared to controls. (C) Ct- CR parasite burden in the fecal samples of CD8 + IEL-transferred Rag2-IL2Rγ-CD47 triple knockout mice. The area under the curve (AUC) in the right panel is pooled data from 2 independent experiments. (D) Ct- CR levels in the ileum on DPI-22. (A-C) Pooled data from 2 independent experiments (N=8-10). (D) Data from 1 experiment. Each dot represents individual mice. Error bars, mean + SEM. **p < 0.01, as calculated by t-test.

Article Snippet: Intermediate layer containing IELs was collected and washed with 1X PBS followed by either flowcytometry analysis or used for CD8α IELs purification using EasySepTM mouse CD8a positive selection kit-II (StemCell Technologies; Cat# 18953).

Techniques: Triple Knockout, Infection

Journal: The Journal of Immunology Author Choice

Article Title: Microcrystalline Tyrosine and Aluminum as Adjuvants in Allergen-Specific Immunotherapy Protect from IgE-Mediated Reactivity in Mouse Models and Act Independently of Inflammasome and TLR Signaling

doi: 10.4049/jimmunol.1800035

Figure Lengend Snippet: T cell response after MCT- or alum-adjuvanted immunization. C57BL/6 mice ( n = 5) were adoptively transferred with OT-I and OT-II cells and immunized with 100 μg OVA on MCT (gray bars) or alum (black bars) 1 and 8 d later (individual mice are indicated). On day 15, spleen cells were analyzed by flow cytometry. ( A ) Dot blots and histograms show SIINFEKL-specific CD8 T cell activation (CD44) and proliferation (H2K b pentamer). Frequencies of IFN-γ–producing and IFN-γ– and TNF-α–double-producing CD8 T cells ( B ) and CD4 T cells ( C ). ( D ) Splenocytes were also restimulated in vitro with MHC class I–restricted OVA aa 257–264 (SIINFEKL), MHC class II–restricted OVA aa 323–339, or OVA protein, and IFN-γ in culture supernatants was measured by ELISA. Mean ± SD of means are illustrated. The experiment is representative of two independent experiments with comparable results. * p < 0.05, ** p < 0.01.

Article Snippet: RAG1-deficient CD8 TCR transgenic OT-I mice were originally from Taconic Biosciences (Ry, Denmark).

Techniques: Flow Cytometry, Activation Assay, In Vitro, Enzyme-linked Immunosorbent Assay