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: Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ 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: Zeta Potential Analyzer, Isolation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

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: BOLT activates T cell PI3K-AKT-mTOR pathway to enhance T cell anti-tumor effect. (A) Flow cytometry plots compare IFN-γ and IL-2 expression at pH 7.8 and 6.0 along with various doses of BOLT in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (B, C) Bar graphs show IFN-γ and IL-2 expression in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (D) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 antibodies and incubated for 3 days with cell culture media of different pH levels. Western blot was performed to determine the phosphorylation of Akt and S6 under acidic conditions (pH 6.5) and alkaline pH (7.8). (E) Activated CD4 + T cells were treated with 0, 0.25, and 0.5 mg/mL doses of BOLT following CD4 + T cells activation at pH 7.8. Western blot analysis showing the phosphorylation of Akt and S6 were observed. (F) CD4 + T cells were activated and treated with BOLT at acidic pH. Western blot analysis was performed to determine the phosphorylation of Akt and S6. Two-way ANOVA was used for multiple comparisons. Experiments were conducted in triplicate. Data are mean ± SEM, ∗ 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: Flow Cytometry, Expressing, Incubation, Cell Culture, Western Blot, Phospho-proteomics, Activation Assay

Journal: STAR Protocols

Article Title: Protocol for potent activation of T cells using BPI-stimulated murine bone marrow-derived cells

doi: 10.1016/j.xpro.2026.104519

Figure Lengend Snippet: Expected results of this protocol Naïve CD4 + T cells cultured for d5 in supernatant of untreated BMDCs (SN NT) or in supernatant of BPI-treated BMDCs (SN BPI). (A) Representative dot blot of flow cytometric analysis of CD62L and CD44 cell surface presentation. (B) IL-22 secretion measured by Luminex technology, n = 4. Data are shown as means ± SEM. Statistical testing was performed using Student`s ratio paired t test.

Article Snippet: Note: If no FACS device is available, the sorting of naïve T cells can be performed using a naïve MACS Sort Kit from Miltenyi Biotec (130-104-453 ).

Techniques: Cell Culture, Dot Blot, Luminex

Journal: STAR Protocols

Article Title: Protocol for potent activation of T cells using BPI-stimulated murine bone marrow-derived cells

doi: 10.1016/j.xpro.2026.104519

Figure Lengend Snippet: Flow cytometric quality control of CD4 + T-cell sort via MACS technique

Article Snippet: Naive CD4 + T-Cell Isolation Kit, mouse , Miltenyi Biotec , Cat# 130-104-453.

Techniques: Control

Journal: STAR Protocols

Article Title: Protocol for potent activation of T cells using BPI-stimulated murine bone marrow-derived cells

doi: 10.1016/j.xpro.2026.104519

Figure Lengend Snippet: Gating strategy for further isolation of CD4 + T-cell populations via fluorescence-activated cell sorting

Article Snippet: Naive CD4 + T-Cell Isolation Kit, mouse , Miltenyi Biotec , Cat# 130-104-453.

Techniques: Isolation, Fluorescence, FACS

Journal: STAR Protocols

Article Title: Protocol for potent activation of T cells using BPI-stimulated murine bone marrow-derived cells

doi: 10.1016/j.xpro.2026.104519

Figure Lengend Snippet: Expected results of this protocol Naïve CD4 + T cells cultured for d5 in supernatant of untreated BMDCs (SN NT) or in supernatant of BPI-treated BMDCs (SN BPI). (A) Representative dot blot of flow cytometric analysis of CD62L and CD44 cell surface presentation. (B) IL-22 secretion measured by Luminex technology, n = 4. Data are shown as means ± SEM. Statistical testing was performed using Student`s ratio paired t test.

Article Snippet: Naive CD4 + T-Cell Isolation Kit, mouse , Miltenyi Biotec , Cat# 130-104-453.

Techniques: Cell Culture, Dot Blot, Luminex

Journal: Cancer Research

Article Title: CCL5 hi CD4 + T Cells Regulate Macrophage Polarization and Promote Immunotherapy Response in Bladder Cancer

doi: 10.1158/0008-5472.CAN-25-2220

Figure Lengend Snippet: CD4 + T-cell diversity is associated with the response to ICI therapy in bladder cancer. A, Correlation heatmap of 22 IC types with treatment outcomes (CR/PR) in the IMvigor210 cohort ( n = 298) and GSE176307 cohort ( n = 89). Asterisks indicate statistically significant correlations (*, P < 0.05). B, Differential abundance of memory activated CD4 + T cells between CR/PR and SD/PD groups. SD, stable disease. C, Differential abundance of memory activated CD4 + T cells between CR and PR groups in the IMvigor210 cohort. D, Kaplan–Meier survival analysis stratified by memory activated CD4 + T-cell infiltration levels in the IMvigor210 cohort. E, Kaplan–Meier survival analysis stratified by memory activated CD4 + T-cell infiltration levels in the GSE176307 cohort. F, Multivariable Cox regression models integrating memory activated CD4 + T-cell abundance with four distinct clinical parameter combinations in the IMvigor210 cohort, showing hazard ratios (HR), 95% confidence intervals (CI), and P values. P values were calculated as follows: Spearman correlation ( A ), two-sided unpaired Wilcoxon rank-sum tests ( B and C ), log-rank test ( D and E ), and Wald tests ( F ).

Article Snippet: To obtain OT-II CCL5 hi -CD4 + T cells, naïve CD4 + T cells were enriched from OT-II mouse spleens using a Naïve CD4 + T Cell Isolation Kit (130-104-453, Miltenyi Biotec).

Techniques:

Journal: Cancer Research

Article Title: CCL5 hi CD4 + T Cells Regulate Macrophage Polarization and Promote Immunotherapy Response in Bladder Cancer

doi: 10.1158/0008-5472.CAN-25-2220

Figure Lengend Snippet: CCL5 hi -CD4 + T-cell subset with memory activation features predicts PD-1/PD-L1 blockade response. A, Schematic representation of scRNA-seq analysis (10x Genomics platform) performed on tumor tissues from five patients in the in-house cohort (Daping cohort 1). BLCA, bladder cancer. B, Uniform Manifold Approximation and Projection (UMAP) plots showing cell types in human bladder cancer tissues. C, UMAP plots showing distinct CD4 + T-cell subsets in human bladder cancer tissues. D, UMAP plots showing memory-activated CD4 + T-cell signature scores across different CD4 + T-cell subsets in human bladder cancer. E, UMAP plots integrating CD4 + T-cell subsets from the in-house cohort (Daping cohort 1, n = 5 samples) and the tumor samples of the PRJNA662018 dataset ( n = 8 samples). F, Heatmap of top differentially expressed marker genes for each CD4 + T-cell subset. G, Difference in the ratio of the CCL5 hi -CD4 + T-cell signature to CD4 expression between CR/PR and stable disease (SD)/PD groups in the IMvigor210 cohort. H, Representative mIHC images and quantification of the proportion of CD4 + CCL5 + cells among total CD4 + T cells in CR ( n = 12) vs. non-CR ( n = 28) patients. CD4, green; CCL5, red; nuclei, DAPI, blue. Scale bars, 40 μm. White arrows, double-positive cells. I, UMAP plots showing two distinct CD4 + T-cell subsets (left) and nonregulatory CD4 + T-cell subsets (right) in murine bladder cancer scRNA-seq data ( n = 9 samples). J, Non-negative least squares (NNLS) regression analysis showing the similarity of CD4 + T-cell subsets between human and murine. K, Representative flow cytometry plots showing differential CCL5 expression frequency between CD25 − CXCR6 + CD4 + T cells and CD25 − CXCR6 − CD4 + T-cell subsets. L, Differential CCL5 expression between CD25 − CXCR6 + and CD25 − CXCR6 − CD4 + T-cell subsets ( n = 7 mice). M, Schematic diagram of MB49 tumor implantation followed by the adoptive transfer of CCL5 hi -CD4 + T cells and anti–PD-1 treatment. N, Change in tumor volumes at multiple time points among different treatment groups ( n = 7 mice/group). P values were calculated as follows: two-sided unpaired Wilcoxon rank-sum test ( G ), two-sided unpaired t test ( H ), two-sided paired t test ( L ), and repeated measures two-way ANOVA ( N ).

Article Snippet: To obtain OT-II CCL5 hi -CD4 + T cells, naïve CD4 + T cells were enriched from OT-II mouse spleens using a Naïve CD4 + T Cell Isolation Kit (130-104-453, Miltenyi Biotec).

Techniques: Activation Assay, Marker, Expressing, Flow Cytometry, Tumor Implantation, Adoptive Transfer Assay

Journal: Cancer Research

Article Title: CCL5 hi CD4 + T Cells Regulate Macrophage Polarization and Promote Immunotherapy Response in Bladder Cancer

doi: 10.1158/0008-5472.CAN-25-2220

Figure Lengend Snippet: CCL5 hi -CD4 + T cells promote M1 macrophage polarization via CCL5/CCR1 signaling to enhance ICI treatment response. A, Heatmap of the top differentially expressed regulons (identified by SCENIC) for each CD4 + T-cell subset. B, Plot of differentially expressed genes, highlighting enrichment in CCL5 hi -CD4 + T cells compared with other CD4 + T-cell subsets. C, CellChat analysis predicting the interaction likelihood between CCL5 hi -CD4 + T cells (source) and myeloid cell subsets (target). Dot size, color intensity, and line width represent the number of interactions. D, Heatmap displaying the activity of different pathway signatures across distinct CD4 + T-cell subsets. E, Heatmap showing the correlation between CD4 + T-cell subsets and IC subpopulations. Correlations with a P > 0.05 are masked. F, Representative mIHC images from CR versus non-CR patients in the Daping cohort, showing CCL5 + CD4 + dual-positive cells and CD68 + CD86 + dual-positive cells. CD4, green; CCL5, red; CD68, magenta; CD86, cyan; nuclei, DAPI, blue. Scale bars, 50 μm. White arrows, CCL5 + CD4 + double-positive cells. Top left and right, consecutive sections from CR patients; bottom left and right, consecutive sections from non-CR patients. G, Correlation between the proportion of CCL5 hi -CD4 (CCL5 + CD4 + ) cells and the proportion of M1 macrophages (CD68 + CD86 + ) based on mIHC. H, Differences in M1 macrophage infiltration across treatment groups ( n = 5–7 mice/group). I, Heatmap showing the activity of various signaling pathways predicted by CellChat for interactions between CCL5 hi -CD4 + T cells and myeloid cell subsets. MIF, macrophage migration inhibitory factor. J, Potential interactions between different CD4 + T-cell subsets and macrophages via the CCL5–CCR1 axis. K, CCR1 expression in TME immune subtypes. L, Schematic of coculture system and pharmacologic intervention in RAW264.7 and CCL5 hi -CD4 + T cells. M, Expression of phenotypical markers (M1) on RAW264.7 cells cocultured with CCL5 hi -CD4 + T cells or CCL5 hi -CD4 + T cells plus BX471. N, Expression of phenotypical markers (M1) on RAW264.7 cells cocultured with CCL5 hi -CD4 + T cells or CCL5 hi -CD4 + T cells plus anti-CCL5 neutralizing antibody. O, Schematic of MB49 tumor implantation followed by the adoptive transfer of CCL5 hi -CD4 + T cells, anti–PD-1, and BX471 treatment. P, Representative flow cytometry plots showing the frequency of M1 macrophages (CD206 - CD86 + F4/80 + ) across treatment groups. Q, Differences in M1 macrophage infiltration across treatment groups ( n = 5 mice/group). R, Change in tumor volumes at multiple time points among different treatment groups ( n = 7 mice/group). P values were calculated as follows: Spearman correlation ( E and G ), one-way ANOVA ( H , M , N , and Q ), and repeated measures two-way ANOVA ( R ). DC, dendritic cell.

Article Snippet: To obtain OT-II CCL5 hi -CD4 + T cells, naïve CD4 + T cells were enriched from OT-II mouse spleens using a Naïve CD4 + T Cell Isolation Kit (130-104-453, Miltenyi Biotec).

Techniques: Activity Assay, Protein-Protein interactions, Migration, Expressing, Tumor Implantation, Adoptive Transfer Assay, Flow Cytometry

Journal: Cancer Research

Article Title: CCL5 hi CD4 + T Cells Regulate Macrophage Polarization and Promote Immunotherapy Response in Bladder Cancer

doi: 10.1158/0008-5472.CAN-25-2220

Figure Lengend Snippet: scRNA-seq analysis reveals that CCR6 hi -CD4 subsets can differentiate into CCL5 hi -CD4 subsets in the TME and are linked to the adverse effects of ICI therapy. A, Schematic representation of the scRNA-seq workflow for parallel processing of primary bladder tumor tissue, metastatic lymph-node tissue, and peripheral blood from one patient with bladder cancer. B, Uniform Manifold Approximation and Projection (UMAP) plots showing distinct CD4 + T-cell subsets. C, The proportion of each subset in CD4 + T cells across samples. D, Trajectory analysis overlaid on CD4 + T-cell subsets, illustrating the differentiation pathways. LN, lymph node; PB, peripheral blood; T, tumor. E, Dynamic gene expression trajectories in CD4 + T cells highlighting changes during differentiation. F, Differential abundance of CCR6 hi -CD4 + T cells between CR/PR and stable disease (SD)/PD groups in the IMvigor210 cohort. G, Representative mIHC images and quantification of the proportion of CD4 + CCR6 + cells among total CD4 + T cells in CR ( n = 12) vs. non-CR ( n = 28) samples. CD4, green; CCR6, red; nuclei, DAPI, blue. Scale bars, 50 μm. White arrows, double-positive cells. H, Correlation of CCL5 hi -CD4 + T-cell infiltration abundance with CCR7 hi -CD4 + T cells and CCR6 hi -CD4 + T cells. I, Schematic workflow for isolation of CD45.1 + CD25 − CCR6 + CD4 + T cells, adoptive transfer into CD45.2 recipients, and longitudinal analysis of CD45.1 + CD4 + T-cell dynamics. J, Representative flow cytometry plots and quantification of the proportion of CCL5 + cells in CD45.1 + CD4 + FOXP3 - cells at days 0, 2, and 6 after transfer ( n = 5/group). P values were calculated as follows: two-sided unpaired Wilcoxon rank-sum test ( F ), two-sided unpaired t test ( G ), Spearman correlation ( H ), and one-way ANOVA ( J ).

Article Snippet: To obtain OT-II CCL5 hi -CD4 + T cells, naïve CD4 + T cells were enriched from OT-II mouse spleens using a Naïve CD4 + T Cell Isolation Kit (130-104-453, Miltenyi Biotec).

Techniques: Gene Expression, Isolation, Adoptive Transfer Assay, Flow Cytometry

Journal: Cancer Research

Article Title: CCL5 hi CD4 + T Cells Regulate Macrophage Polarization and Promote Immunotherapy Response in Bladder Cancer

doi: 10.1158/0008-5472.CAN-25-2220

Figure Lengend Snippet: Tumor-derived PGE2 inhibits CCL5 hi -CD4 + T-cell infiltration and promotes resistance to ICI therapy. A, Significantly enriched pathways in high-scoring tumors compared with low-scoring tumors, based on Gene Ontology databases. FC, fold change. B, Significant ligand–receptor pairs between CCR6 hi -CD4 + T cells and tumors of different scores. C, Proportions of CR/PR vs. stable disease (SD)/PD in the top 20% and bottom 20% PGE2 score groups of patients in IMvigor210. D, PGE2 scores stratified by CCR6 hi -CD4 + T-cell and CCL5 hi -CD4 + T-cell abundance. E, ELISA demonstrating the difference in PGE2 levels between supernatants of COX2 WT and COX2 KD2 tumors. F, Differences in the proportion of CCR6 + CCL5 − cells and CCR6 − CCL5 + cells within CD25 − CD4 + T cells across treatment groups ( n = 5/group). G, Differences in the proportion of M1 macrophages (F4/80 + CD206 − CD86 + ) across treatment groups ( n = 5/group). H, Change in tumor volumes at multiple time points among different treatment groups ( n = 5 mice/group). I, qPCR analysis of CCL5 gene expression in CCR6 hi -CD4 + T cells after culture under different conditions. J, Proportion of CCL5 + cells within CD45.1 + CD4 + FOXP3 − T cells at indicated time points after the adoptive transfer of CD45.1 + CD25 − CCR6 + CD4 + T cells into COX2 WT , COX2 KD2 , and COX2 WT + PGE2 groups. K, Change in tumor volumes at multiple time points among different treatment groups ( n = 5 mice/group). L, Change in tumor volumes at multiple time points among different treatment groups ( n = 5 mice/group). M, Differences in the proportion of M1 macrophages across treatment groups in L ( n = 5 mice/group). N, Schematic of MB49 tumor implantation followed by treatment with anti–PD-1 and celecoxib. O, Change in tumor volumes at multiple time points among different treatment groups ( n = 7 mice/group). P, Differences in CCL5 hi -CD4 + T-cell infiltration across treatment groups ( n = 5 mice/group). P values were calculated as follows: Fisher exact test ( C ), two-sided unpaired Wilcoxon rank-sum test ( D ), two-sided unpaired t test ( E ), one-way ANOVA ( F , G , I , J , M , and P ), and repeated measures two-way ANOVA ( H , K , L , and O ).

Article Snippet: To obtain OT-II CCL5 hi -CD4 + T cells, naïve CD4 + T cells were enriched from OT-II mouse spleens using a Naïve CD4 + T Cell Isolation Kit (130-104-453, Miltenyi Biotec).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Adoptive Transfer Assay, Tumor Implantation

Journal: medRxiv

Article Title: Vδ2 T cell activation by malaria is enhanced in second infection via the cell extrinsic cytokine milieu

doi: 10.64898/2026.04.29.26352021

Figure Lengend Snippet: A ) Vδ2 and Vδ1 T cell expression of HLA-DR and CD86 during CHMI in PBMCs at infection (I, 0 days post infection (d.p.i.)), treatment (T, 8 d.p.i.), 7 (T+7, 15 d.p.i.) and 21 (T+21, 29 d.p.i.) days post-treatment ( n = 12). ( B ) HLA-DR, CD86 and CD40 expression on Vδ2 T cells from malaria-naïve donors ( n = 15) ex vivo and after culture for 5 days with media alone, HMBPP, uRBC lysate and pRBC lysate. ( C ) Activation of isolated naïve CD4 T cells with γδ T cells ( n = 6) isolated ex vivo , or after 3-day culture with media, uRBC lysate or pRBC lysate. Positive control is CD4 T cells with CD3/CD28 beads and IL-12, IL-23 and TGF-β (B+C). Frequency of divided cellS in CD4 T cell population after co-culture is shown. ( D ) Phagocytosis of uRBC or pRBC stained with Cell Trace Violet in Vδ2 T cells (n=6) from PBMCs at T+7 during CHMI. Centre lines represent median, box limits indicate the upper and lower quartiles, whiskers extend to 1.5 times the interquartile range. Lines represent paired observations. P from paired data are Wilcoxon signed rank tests. Unpaired comparisons made with Mann-Whitney U test. See also Supplementary Figures 6-9.

Article Snippet: For the mixed lymphocyte reaction assay of γδ and naive CD4 T cells, cells were isolated from PBMCs using the TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec; Cat. #130-092-892), Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec; Cat. #130- 094-131), a QuadroMACST M Separator (Miltenyi Biotec; Cat. #130-090-976), pre-Separation Filters (Miltenyi Biotec; Cat. #130-041-407) and LS Colums (Miltenyi Biotec; Cat. #130-042- 401) as per manufacturer’s instructions. γδ T cells were isolated from malaria-naïve PBMCs (ARC) co-cultured with blank media, 10 6 uRBCs or 10 6 pRBCs for 3 days (ensure high effector cell viability) at 37°C, 5% CO 2 in R10.

Techniques: Expressing, Infection, Ex Vivo, Activation Assay, Isolation, Positive Control, Co-Culture Assay, Staining, MANN-WHITNEY