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:

Article Title: A Mage3/Heat Shock Protein70 DNA Vaccine Induces both Innate and Adaptive Immune Responses for the Antitumor Activity

doi: 10.1016/j.vaccine.2009.09.119

Figure Lengend Snippet: Mice were immunized i.m. with 100 μg of different DNA constructs twice at a two-week interval. Data were mean +/− s.d. of triplicate wells. A, Splenocytes were harvested and incubated with TC-1/Mage3 cell lysate for 20 hr. The frequency of T cells producing IFN-γ was determined by using Elispot. B, Specificity of splenocytes from pc-sMage3Hsp-immunized mice responding to TC-1/Mage3 vs. parental TC-1 cells, and Mage3 protein vs. irrelevant CD20 protein was determined by using Elispot. C, Response of CD4+ T cells to Mage3-pulsed DCs was determined by using Elispot. D, CTL response of splenocytes to TC-1/Mage3 was determined by using a 51Cr-release assay.

Article Snippet: Six- to eight-week-old female C57BL/6 mice, CD4 + knockout (KO) mice, and CD8 + KO mice were purchased from Harlan Sprague Dawley, Taconic, and Jackson Laboratory, respectively.

Techniques: Construct, Incubation, Enzyme-linked Immunospot, Release Assay

Journal:

Article Title: A Mage3/Heat Shock Protein70 DNA Vaccine Induces both Innate and Adaptive Immune Responses for the Antitumor Activity

doi: 10.1016/j.vaccine.2009.09.119

Figure Lengend Snippet: A, Expression and secretion of Mage3 from pc-Mage3 or pc-sMage3-transfected 293 T cells, tested by western blotting (the upper panel) or ELISA (the lower panel). B. Antibody reponse from pc-Mage3 or pc-sMage3 immunized mice evaluated by ELISA. C. CD4+ and CD8+ T cell responses from pc-Mage3 or pc-sMage3 immunized mice evaluated by intracellular IFN-γ staining. The data are the representative of the two independent experiments.

Article Snippet: Six- to eight-week-old female C57BL/6 mice, CD4 + knockout (KO) mice, and CD8 + KO mice were purchased from Harlan Sprague Dawley, Taconic, and Jackson Laboratory, respectively.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal:

Article Title: A Mage3/Heat Shock Protein70 DNA Vaccine Induces both Innate and Adaptive Immune Responses for the Antitumor Activity

doi: 10.1016/j.vaccine.2009.09.119

Figure Lengend Snippet: A C57BL/6 wild-type, CD4+ KO, or CD8+ KO mice were inoculated with newly generated TC-1/Mage3 cells. Three days later, the mice were immunized by i.m. injection with 100 μg of pc-sMage3Hsp DNA or PBS twice at a one-week interval.. The percentage of tumor-free mice was calculated on the indicated days. B. C57BL/6 wild-type, CD4+ KO, or CD8+ KO mice were immunized by i.m. injection with 100 μg of pc-sMage3Hsp DNA twice at a two-weeks interval. Two weeks after the second injection, the mice were inoculated s.c. with in vitro maintained TC-1/Mage3 tumor cells. The percentage of tumor-free mice was calculated on the indicated days. C. C57BL/6 wild-type and CD8+ KO mice were immunized by i.m. injection with 100 μg of pc-Mage3Hsp DNA twice at a two-week interval. Two weeks after the second injection, the mice were inoculated s.c. with TC-1/Mage3 tumor cells. The CD8+ KO mice and a half of the C57BL/6 wild-type mice received PK136 treatment prior to and after TC-1/Mage3 challenge. The remaining half of wild-type mice received control IgG administration. The percentage of tumor-free mice was calculated on the indicated days. D. C57BL/6 mice were immunized by i.m. injection with 100 g of pc-Mage3Hsp DNA twice at a two-week interval. Two weeks after the second injection, the mice were inoculated s.c. with TC-1 tumor cells. Half of mice received PK136 treatment, and half received control IgG administration. The percentage of tumor-free mice was calculated on the indicated days. E. C57BL/6 mice were immunized by i.m. injection with 100 μg of pc-sHsp, pc-Mage3Hsp DNA or PBS twice at a two-week interval. Two weeks after the second injection, the mice were inoculated s.c. with the newly generated TC-1/Mage3 tumor cells. Half of pc-sHsp-immunized mice and all pc-sMage3Hsp-immunized mice received PK136 treatment. The percentage of tumor-free mice was calculated on the indicated days.

Article Snippet: Six- to eight-week-old female C57BL/6 mice, CD4 + knockout (KO) mice, and CD8 + KO mice were purchased from Harlan Sprague Dawley, Taconic, and Jackson Laboratory, respectively.

Techniques: Generated, Injection, In Vitro, Control

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: SLC1A5 is linked to T cell exhaustion and upregulated following L-ky administration. A Mouse CD8 + T cells were activated through CD3/CD28/IL-2 stimulation, followed by chronic stimulation to induce T cell exhaustion. TPEX (PD1 + TIM3 − ) and TEX (PD1 + TIM3 + ) cells were then sorted with FACS for RNA-seq analysis. B - C Volcano plots ( B ) and heatmap ( C ) for DEGs between TPEX and TEX identified using RNA-seq analysis. D FACS analysis of the MFI of SLC1A5 in TEX and TPEX. E–F. qPCR ( E ) and WB ( F ) analysis of SLC1A5 mRNA and protein levels in TEX and TPEX. L-ky was supplemented to culture system of T cells. FACS analysis of the PD1 + TIM3. + CD8 T cell population after L-ky treatment. I - J qPCR ( I ) and WB ( J ) analysis of SLC1A5 mRNA and protein levels in cells after L-ky treatment. K Immunofluorescence detection of nuclear translocation of AHR in T cells after L-ky treatment. L ChIP-qPCR analysis of AHR binding to the SLC1A5 promoter using anti-AHR antibody. Cell-based experiments repeated six times. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: RNA Sequencing, Immunofluorescence, Translocation Assay, ChIP-qPCR, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: SLC1A5 knockout diminishes L-ky-mediated T cell exhaustion in mice. A Mouse LLC cells overexpressing IDO1 or not implanted into either WT or SLC1A5-/- mice subcutaneously, followed by animal sacrifice on day 28. B Growth rate of subcutaneous tumors in mice. C Diagram and weight of tumors formed by LLC cells on day 28. D FACS analysis of the number of CD45 + CD8 + T cells in tumors formed by LLC cells. E FACS analysis of the number of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3 − ) cells in tumors formed by LLC cells. F FACS analysis of the number of SLC1A5 + PD-1 + CD8 + T cells in tumors formed by LLC cells. G FACS analysis of the number of GZMB + CD8 + T cells in tumors formed by LLC cells. H CD8 + T cells from either WT or SLC1A5-/- mice were extracted, activated by CD3/CD28 stimulation, labeled with CFSE, and then treated with L-ky. Groups included: 'T activated' (stimulation in pure medium, system control), 'WT + PBS' (stimulation with vehicle, experimental control), and 'WT + L-Ky' (treatment). Note that statistical comparisons for the L-Ky effect were performed against the WT + PBS vehicle control to account for handling variations. I FACS analysis of CFSE-positive cells to assess T cell proliferation. J WT mice bearing IDO1-overexpressing LLC cells were subjected to tryptophan-deprivation diets or normal diets. K Analysis of LLC cell growth rate in mice after tryptophan deprivation. L Weight of tumors formed by LLC cells after tryptophan deprivation. M FACS analysis of the number of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3. − ) cells in tumors formed by LLC cells after tryptophan deprivation. Each group contained 6 mice. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: Knock-Out, Labeling, Control

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: SLC1A5 knockout in T cells inhibits LLC cell tumorigenesis in mice. A CD8 + T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice were generated, to which IDO1-overexpressing LLC cells were implanted. B Growth rate of subcutaneous tumors formed by LLC cells. C Diagram and weight of subcutaneous tumors formed by LLC cells on day 28. D FACS analysis of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3 − ) in LLC-formed tumors. E FACS analysis of GZMB + CD8 + T cells in LLC-formed tumors. F Immunofluorescence detection of AHR nuclear translocation in CD8 + T cells within tumor tissue. G CD8 + T cells were isolated from tumor tissues in SLC1A5 cko mice and SLC1A5 fl/fl mice, followed by the treatment of 14C-labeled kynurenine. H Uptake of 14C-labeled kynurenine by T cells. I Quantification of kynurenine metabolites (14C-3-HK and KYNA) in T cells. J CD8 + T cells extracted from SLC1A5 cko mice were treated with the AHR agonist 2, followed by FACS analysis of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3 − ) cells. L ELISA measurement of IFN-γ, GZMB, and TNF-α production by CD8. + T cells. Animal experiments included six mice per group, and cell-based experiments repeated six times. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: Knock-Out, Generated, Immunofluorescence, Translocation Assay, Isolation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: L-ky enhances chromatin accessibility in T cells. A ATAC-seq analysis illustrating changes in chromatin accessibility in in vitro chronically activated CD8 + T cells following 48 h of L-ky treatment (100 uM). B Enrichment analysis of differentially accessible regions for genes significantly affected by L-ky treatment. C GO enrichment analysis of genes near differentially accessible regions. D - E ChIP-seq analysis using H3K27ac and H3K9ac antibodies to assess the occupancy of these modifications in CD8. + T cells after L-ky treatment. F - G Distribution of H3K27ac and H3K9ac modifications across the genome following L-ky treatment. H Integrative Genomics Viewer analysis of H3K27ac and H3K9ac occupancy in the 2–3 kb upstream region of SLC1A5, PD-1, TIM3, and CTLA4 promoters. I ChIP analysis using H3K27ac and H3K9ac antibodies, followed by qPCR to quantify enrichment of these modifications at the SLC1A5, PD-1, TIM3, and CTLA4 promoters. Cell-based experiments repeated six times. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: In Vitro, ChIP-sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: AHR recruits chromatin modifying enzyme FANCD2 to enhance SLC1A5 expression. A An APEX-AHR fusion protein was constructed and transfected into T cells. Biotin-phenol and H 2 O 2 were then added, followed by streptavidin-mediated IP and mass spectrometry to identify AHR-interacting proteins, with chromatin-related proteins filtered using the BIOGRID database. B A GST-AHR fusion vector was constructed, loaded in T cells, and subjected to affinity chromatography using GSH. Enriched products were analyzed via SDS-PAGE. C - D HA-AHR and Flag-FANCD2 vectors were transfected into HEK293T cells. IP with anti-HA or anti-Flag was followed by WB analysis to detect Flag and HA expression. E HA-AHR was loaded in T cells treated with varying L-ky doses. IP with anti-HA antibody was followed by WB analysis to detect AHR and FANCD2 expression. F ChIP analysis using anti-FANCD2 assessed FANCD2 enrichment at the SLC1A5, PD-1, TIM3, and CTLA4 promoters via qPCR. G L-ky-treated T cells were treated with FEN1-IN-1. H - I qPCR and WB analysis of SLC1A5 mRNA and protein expression in T cells following L-ky and FEN1-IN-1 treatments. J ELISA analysis of IFN-γ, GZMB, and TNF-α secretion by T cells following L-ky and FEN1-IN-1 treatments. Cell-based experiments repeated six times. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: Expressing, Construct, Transfection, Mass Spectrometry, Plasmid Preparation, Affinity Chromatography, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: Targeting FANCD2 restores T cell function. A WT mice implanted with IDO1-overexpressing LLC cells were further treated with the FANCD2 antagonist TNG463. B Growth rate of LLC cells in vivo. C Diagram and weight of LLC-formed tumors on day 28. D FACS analysis of the number of SLC1A5 + CD8 + T cells in tumor tissues. E FACS analysis of the number of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3 − ) cells in LLC-induced tumors. F - G FACS analysis of the number of GZMB + CD8 + (F) and TNFA + CD8 + (G) T cells in tumor tissues. H IHC of C-Cas-3 staining in tumor tissues. I CD8 + T cells treated with L-ky alone or alongside TNG463 were co-cultured with LLC cells. J LDH release from LLC cells after co-culture with T cells, measured by LDH assay kit. K FACS analysis of CFSE. + T cell counts. Animal experiments involved six mice per group, with cell-based experiments repeated six times. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: Cell Function Assay, In Vivo, Staining, Cell Culture, Co-Culture Assay, Lactate Dehydrogenase Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: SLC1A5 knockout in T cells enhances the therapeutic efficacy of anti-PD1 in mice. A IDO1-overexpressing LLC cells were injected into SLC1A5 fl/fl or SLC1A5 CKO mice through the tail vein, followed by treatment with anti-PD-1 monoclonal antibody (mAb) or control IgG. B Number of metastatic nodules formed by LLC cells in the lungs of mice. C Kaplan–Meier survival analysis of the mice. D GFP-IDO1-overexpressing LLC cells were implanted into C57BL/6 mice, followed by treatment with V-9302 (an SLC1A5 inhibitor), anti-PD-1 mAb, or a combination of both. E – F Analysis of bioluminescence intensity in the mice at 7, 14, and 28 d post-treatment. G-H. FACS analysis of the number of GZMB + CD8 + ( G ) and TNFA + CD8 + ( H ) T cells in tumor tissues. I FACS analysis of the number of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3. − ) cells in LLC-induced tumors. J Kaplan–Meier survival analysis of the mice. Animal experiments involved six mice per group. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: The animal models used in this study included C57BL/6 wild-type (WT) mice, SLC1A5 knockout mice (SLC1A5 −/− ), SLC1A5 flox/flox (SLC1A5 fl/fl ) mice, CD8-Cre transgenic mice, and CD8+ T cell-specific SLC1A5 conditional knockout (SLC1A5 cko ) mice, all purchased from Charles River (China).

Techniques: Knock-Out, Drug discovery, Injection, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Depletion of conventional CD4+ T cells is required for robust priming and dissemination of tumor antigen-specific CD8+ T cells in the setting of anti-CD4 therapy

doi: 10.1136/jitc-2024-010170

Figure Lengend Snippet: Removal of Ag-specific CD4 + T cells is required to induce systemic CD8 + T cell responses against tumor antigens. ( A ) Congenically marked Thy1.1 + pmel cells were transferred into Foxp3-DTR and WT B6 mice 1 day prior to tumor inoculation. WT mice were either untreated or treated with anti-CD4 on days 4 and 10 after tumor inoculation. Foxp3-DTR mice were treated for five consecutive days with DT beginning on day four post tumor injection. Tissues were harvested for flow cytometry analysis on day 12 after tumor inoculation. ( B ) Proportion of Thy1.1 + pmel cells; gated on live CD8 + T cells across tissues on day 12. ( C ) Proportion of total CD8 + T cells, gated on live CD45 + lymphocytes in skin and tumor on day 12. ( D ) RAG knockout mice were reconstituted with polyclonal CD8 + T cells and congenically marked pmel cells along with either polyclonal CD4 + T cells or OTII CD4 + T cells 1 day prior to tumor implantation. Tumors were left to grow for 12 days prior to analyzing pmel cell priming and dissemination by flow cytometry. ( E ) Proportion of CD44 hi Thy1.1 + (TDLN, Spleen, Skin) or CD44 hi (tumor) pmel cells, gated on live CD8 + T cells on day 12. ( F ) Proportion of total CD8+T cells, gated on live lymphocytes in skin and tumor on day 12. Experiments were repeated twice with n≥4 mice per group. Data are pooled from two independent experiments with similar results. Flow plots depict representative mice; bars represent means and error bars represent SEM One-way ANOVAs with multiple comparisons for experiments with three groups, and paired t-tests for experiments with two groups were used to determine statistical significance. ANOVA, analysis of variance; DT, diphtheria toxin; TDLN, tumor draining lymph node; WT, wild-type; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: For experiments involving WT versus OTII CD4 + T cell reconstitution in RAG knockout mice, biotin positive selection kits (STEMCELL #17655) were used to isolate total CD4 + T cells from naïve C57BL/6 or OTII donor mice and total T cell (STEMCELL #19851) or CD8+T cell (STEMCELL #19853) isolation kits were used to isolate total CD8 T cells from naïve C57BL/6 donor mice.

Techniques: Injection, Flow Cytometry, Knock-Out, Tumor Implantation

Journal: Journal for Immunotherapy of Cancer

Article Title: Depletion of conventional CD4+ T cells is required for robust priming and dissemination of tumor antigen-specific CD8+ T cells in the setting of anti-CD4 therapy

doi: 10.1136/jitc-2024-010170

Figure Lengend Snippet: cDC1-dependent priming of tumor-specific CD8 + T cells does not require depletion of CD4-expressing antigen-presenting cells. WT B6 mice were either untreated or treated with anti-CD4 on days 4 and 10 post intradermal B16 tumor implantation. TDLNs were digested and assessed by flow cytometry to identify various myeloid cell populations ( A–D ). ( A ) Flow plots of CD4 + non-T cells in WT tdLNs on day 12. ( B ) Flow plots showing cDC1 and cDC2s in tdLNs on day 12. Populations are gated out of live singlets, lymphocytes, F480 − /CD19 − cells, CD11b ± CD11c +/− , MHCII + Ly6C − cells. Xcr1 + Sirp1a − cells are cDC1s and Xcr1 − Sirp1a + cells are cDC2s. ( C ) Proportion of and total cDC2s per tdLN on day 12. ( D ) Proportion and total of cDC1s per tdLN on day 12. CD86 expression on cDC1s on day 12. ( E ) Mice were treated as described in . Normalized (relative to no treat) number of cDC1s per TDLN and CD86 expression on cDC1s in TDLNs from untreated, anti-CD4 treated and dual ICB-treated mice. ( F ) Congenically marked pmel cells were transferred into WT B6 and Batf3 knockout mice 1 day prior to B16 implantation. Mice were treated with anti-CD4 on days 4 and 10 post tumor injection and then tissues were harvested on day 12 to assess priming by flow cytometry. Proportions CD44 hi Thy1.1 + pmel cells out of CD8 + T cells across tissues in WT and Batf3 KO mice on day 12. ( G ) Naïve, congenically marked pmel cells were transferred into RAG knockout mice lacking CD4 + T cells. For two groups, intradermal B16 tumors were implanted 1 day later. Anti-CD4 treatment was given to deplete CD4 + non-APCs 4 days after tumor implantation. ( G ) Pmel cells across tissues on day 12 from RAG −/− mice reconstituted with pmel cells only. Experiments were repeated at least two times with similar results and n≥3 mice per group. Flow plots depict representative mice; bars represent means and error bars represent SEM. One-way ANOVA with multiple comparisons was used to determine statistical significance for experiments with more than two groups. Paired t-test was used to determine statistical significance in experiments with two groups. n.s. indicates p>0.05. ANOVA, analysis of variance; cDC2s, conventional type-2 dendritic cells; ICB, immune checkpoint blockade; TDLN, tumor draining lymph node; WT, wild-type; *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: For experiments involving WT versus OTII CD4 + T cell reconstitution in RAG knockout mice, biotin positive selection kits (STEMCELL #17655) were used to isolate total CD4 + T cells from naïve C57BL/6 or OTII donor mice and total T cell (STEMCELL #19851) or CD8+T cell (STEMCELL #19853) isolation kits were used to isolate total CD8 T cells from naïve C57BL/6 donor mice.

Techniques: Expressing, Tumor Implantation, Flow Cytometry, Knock-Out, Injection

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