cd8a microbeads  (Miltenyi Biotec)

Journal: iScience

Article Title: CCL signaling drives T Cell–Macrophage crosstalk in the mouse colon during chronic Trypanosoma cruzi infection

doi: 10.1016/j.isci.2026.116611

Figure Lengend Snippet: CD8 + T cytotoxic cells are the predominant subset within the T and NK cell compartment in the colon of chronically infected mice (A) Uniform manifold approximation and projection (UMAP) plot showing nine T and NK cell subclusters. Each point represents a single cell. (B) Bar graph depicts the relative percentages of each cell subtype in naive and infected mice. (C) Representative flow cytometry plots of CD8 + T cells in naive (left) and infected mice (right). Cells were gated on live, singlets, CD3 + cells. (D) Violin plot shows the percentage of CD8 + T cells in naive and infected mice. (E) Representative flow cytometry plots of T follicular helper cells in naive (left) and infected mice (right). Cells were gated on live, singlets, CD3 + CD4 + cells. (F) Violin plot shows the percentage of PD-1 + CXCR4 + T follicular helper cells in naive and chronically infected mice. Data are representative of two independent experiments with a total of 6 (in D) or 4 (in F) mice per group. In (D and F), violin plots show the distribution of values for each group. Individual points represent biological replicates. Large dashed horizontal lines indicate the median. Statistical significance was determined by an unpaired t test. ∗∗∗: p < 0.001; ns: not significant.

Article Snippet: CD8a (Ly-2) Microbeads , Miltenyi Biotec , Cat-130-117-044.

Techniques: Infection, Single Cell, Flow Cytometry

Journal: iScience

Article Title: CCL signaling drives T Cell–Macrophage crosstalk in the mouse colon during chronic Trypanosoma cruzi infection

doi: 10.1016/j.isci.2026.116611

Figure Lengend Snippet: CD8 + cytotoxic T cells contribute as central mediators within the CCL signaling network (A) Violin plots show the distribution and density of CCL5 (left) and CCR5 (right) expression across T and NK cell subsets, as well as macrophages, in the colon of naive and chronically infected mice. Wider sections of the violin indicate a higher proportion of cells expressing CCL5 or CCR5 at that level. Data were generated from CellChat-guided analysis of scRNA-seq profiles. (B) Representative flow cytometry plots show CD8 + CCR5 + T cells in the colon of naive (upper panel) and chronically infected mice (bottom panel). Cells were gated on live, singlets, and CD3 + cells. (C) Representative flow cytometry plots show F4/80 + CCR5 + T cells in naive (upper panel) and chronically infected mice (bottom panel). Cells were gated on live, singlets, and CD45 + cells. (D) Violin plot shows the frequency of CD8 + CCR5 + T cells in naive and chronically infected mice. Data are representative of two independent experiments ( n = 4 mice per group). (E) mRNA quantification of Ccl5 and Ccr5 by qRT-PCR in naive and chronically infected mice. The plot shows log 2 fold changes (FC) in gene expression determined by the ΔC t method with normalization to GAPDH. The horizontal line indicates the median value for each group, with individual data points plotted. CD8 + T cells were purified from the lamina propria of naive and chronically infected mice using magnetic bead–based separation prior to RNA extraction. Data are from three independent experiments. (F) Violin plot shows the frequency of F4/80 + CCR5 + T cells in the colon of naive and chronically infected mice. Data are representative of two independent experiments ( n = 3 mice per group). In (D and F), violin plots show the distribution of values for each group. Individual points represent biological replicates. Large dashed horizontal lines indicate the median. Statistical significance was determined by an unpaired t test. ∗∗: p < 0.01; ns: not significant.

Article Snippet: CD8a (Ly-2) Microbeads , Miltenyi Biotec , Cat-130-117-044.

Techniques: Expressing, Infection, Generated, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Purification, RNA Extraction

Journal: iScience

Article Title: CCL signaling drives T Cell–Macrophage crosstalk in the mouse colon during chronic Trypanosoma cruzi infection

doi: 10.1016/j.isci.2026.116611

Figure Lengend Snippet: CD8 + cytotoxic T cell–macrophage interactions likely shape colonic immune homeostasis during chronic T. cruzi infection (A) Volcano plot showing differentially expressed genes (DEGs) in lamina propria CD8 + cytotoxic T cells from infected mice compared with naive controls. Red dots depict upregulated genes, blue dots indicate downregulated genes, and gray dots represent genes with a non-significant change in expression. Genes with log 2 fold change ≥1.5, and p < 0.05 were considered significant. (B) Top five differentially regulated canonical pathways in lamina propria CD8 + cytotoxic T cells from infected mice, as predicted by ingenuity pathway analysis (IPA). Bars represent −log ( p value); red indicates pathway activation and blue indicates pathway inhibition. (C) Top 10 upregulated genes associated with the IL-4/IL-13 signaling pathway (left) and the pathogen-induced cytokine storm (PICS) pathway (right) in CD8 + cytotoxic T cells. (D) Volcano plot of DEGs in lamina propria macrophages from infected mice compared with naive controls, displayed as in (A). (E) Top five differentially regulated canonical pathways in lamina propria macrophages from infected mice, as predicted by IPA, displayed as in (B). (F) Top 10 upregulated genes associated with the S100 protein family signaling pathway (left) and the neutrophil degranulation pathway (right) in macrophages. In (C and F), bars indicate log 2 fold change between infected and naive mice.

Article Snippet: CD8a (Ly-2) Microbeads , Miltenyi Biotec , Cat-130-117-044.

Techniques: Infection, Expressing, Activation Assay, Inhibition

Journal: iScience

Article Title: CCL signaling drives T Cell–Macrophage crosstalk in the mouse colon during chronic Trypanosoma cruzi infection

doi: 10.1016/j.isci.2026.116611

Figure Lengend Snippet: Proposed model of the immune response mechanisms to Trypanosoma cruzi in the mouse colon, integrating our findings with prior studies ① Infected smooth muscle cells release trypomastigotes into the surrounding colonic tissue. ② Trypomastigotes are phagocytosed by macrophages. ③ After 18–24 hs post-internalization, amastigotes are released into the cytoplasm, where they replicate and synthesize proteins that are presented on the macrophage surface. ④ CD8 + and CD4 + T cells recognize T. cruzi peptides presented by MHC complexes. ⑤ Upon activation, CD8 + T cells secrete granzyme A and multiple cytokines. ⑥ Granzyme A -containing granules may enter parasite-infected smooth muscle cells through pores formed by perforin-1 (PRF1; secreted by NK cells), contributing to cell death. ⑦ Tumor necrosis factor (TNF) may promote macrophage polarization to an M1 phenotype by inducing the expression of inducible nitric oxide synthase (NOS2). This enzyme subsequently oxidizes arginine to produce nitric oxide, which may contribute to parasite killing. ⑧ Activated CD4 + T cells can differentiate into Th and Tfh cells that produce Interleukin-4 (IL-4) and Interleukin-13 (IL-13) [⑨], which in turn induce M2 macrophage polarization. ⑩ C-C Motif Ligand 5 (CCL5) could further support M2 macrophage polarization via arginase I (ARG1) upregulation, promoting polyamines associated with parasite multiplication and persistence. ⑪ Amastigotes differentiate into trypomastigotes, completing the cycle by infecting naive smooth muscle cells.

Article Snippet: CD8a (Ly-2) Microbeads , Miltenyi Biotec , Cat-130-117-044.

Techniques: Infection, Activation Assay, Expressing

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl CD4 Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Knock-Out, Generated, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 fl/fl CD4 Cre mice exhibit improved anti-tumor mmune responses. (A-C) Naïve CD4 + T cells were isolated from CD4 Cre or GPCR68 fl/fl CD4 Cre mice and activated using anti-CD3 and anti-CD28 using the culture media under physiologic neutral pH (7.4) or varying pH 6.0, 6.5, or 7.8. Flow cytometry plots showing the expression of IFN-γ and IL-2 in CD4 + T cells from CD4 Cre and GPCR68 fl/fl CD4 Cre mice. Each panel represents the frequency of IFN-γ + and IL-2 + cells. (B) Bar graph summarizing the percentage of IFN-γ + CD4 + T cells at each pH level for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (C) Bar graph showing the percentage of IL-2 + CD4 + T cells at each pH for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (D) Experimental timeline depicting tumor induction and treatment protocol in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (E) Tumor growth curves in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (F) Tumor weight in CD4 Cre versus GPCR68 fl/fl CD4 Cre mice at the time of harvesting on day 21. (G) Representative images of excised tumors at day 21. (H) Flow cytometric analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (I) Flow cytometric analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Flow Cytometry, Expressing, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Anti-tumor effects of borate bioactive glass (BOLT) in B16 tumor. (A) Schematic illustration depicting the induction of B16 melanoma tumors, followed by treatment with BOLT at various time points, and tumor harvesting for subsequent analysis. (B) Tumor growth curves showing tumor volume in Control and BOLT-treated B16 melanoma tumors in mice. (C) Tumor weight at the time of harvesting in the BOLT-treated group compared to the Control. (D) Representative images of excised tumors from Control and BOLT-treated mice. (E) In vivo imaging of tumor-bearing mice in both the Control and BOLT-treated groups. (F) Flow cytometry analysis showing IFN-γ production in CD4 + and CD8 + T cells following BOLT treatment compared to Control. (G) Flow cytometry analysis demonstrated TNF-α production in CD4 + and CD8 + T cells in the BOLT-treated group, with a significant increase observed in CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data represent the mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Control, In Vivo Imaging, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Combinational treatment of BOLT and anti-CTLA-4 blockade enhances anti-tumor immune response in B16 melanoma. (A) C57BL/6 mice were subcutaneously injected with 1 × 10 5 B16 melanoma cells on day 0 to induce tumors. On day 7, mice were randomly divided into groups and treated with either BOLT alone (intratumoral injection administered on alternate days starting from day 7), anti-CTLA-4 (intraperitoneal injection administered on days 9, 11, 13, and 15), or a combination of both treatments. PBS was used as a vehicle Control, while IgG was used as anti-CTLA-4 Control. Tumor growth was monitored throughout the treatment period, and tumors were harvested for analysis on day 21. (B-C) Tumor growth curves and area under the curve (AUC) analysis for WT mice treated with BOLT, with or without anti-CTLA-4 antibody, following subcutaneous injection of B16 melanoma cells. Tumor growth was monitored, and analysis was conducted on day 21. (D) Representative images of excised tumors at day 21, showed reduced tumor size in combination-treated mice. (E, F) Flow cytometry analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (G, H) Flow cytometry analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Injection, Control, Flow Cytometry