pe anti human cd8a  (Elabscience Biotechnology)

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Kaplan-Meier curve of GBM patients on standard of care (SOC; red, median=12.4 months) or combinations of SCO with metformin (blue, media=13 months) or DPP4 inhibitors (DPP4i, median=15.9 months) based on the SEER dataset. (B) DPP4 expression from immune lineages as determined by the scRNA-seq of 13 brain tumour patients (3 Grade II; 1 Grade III-IV; 9 GBM). Mean fluorescence intensity (MFI) of DPP-4 from (C) tumor-infiltrating and (D) splenic immune populations. n=5; *** p<0.001 by two-way ANOVA. (E) Representative gating strategy used to define effector, progenitor exhausted (PEX), or terminally exhausted (TEX) populations. (F) DPP-4 MFI from effector, PEX, and TEX populations. n=8; ** p<0.01 by two-way ANOVA. (G) Exhausted CD8 + T cells from GBM and tumor-adjacent tissue were defined as: CD45 + CD11b - CD3 + PD1 + TIGIT + . DPP-4 MFI from TOX + and TOX - population. n=4; * p<0.05 by paired t-test.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Gating strategy to identify immune populations from healthy human PBMCs. (B) DPP-4 geometric MFI in memory CD8 + T cells from healthy donors. n=4; * p<0.05, ** p<0.01, *** p<0.001 by ordinary one-way ANOVA. (C) Diagram showing how naïve CD45.1 + p14 CD8 + T cells were adoptively transferred into recipient wild-type CD45.2 + mice. Recipient mice were infected with 2 x 10 5 plaque forming units (PFU) of LCMV Armstrong intraperitoneally (i.p.) or with 4 x 10 6 LCMV clone 13 intravenously (i.v.) the day after. Peripheral blood was collected on day 16 and 21 to confirm infection, and spleens collected on day 35 for endpoint immune analysis . (D) DPP-4 MFI from splenic CD8 + T cells infected with two different LCMV strains to promote effector, activated, exhausted, and memory differentiation. Clone 13, n=11, Amstrong, n=4; * p<0.05, **** p<0.0001, ns=not significant by one-way ANOVA. (E) Splenic CD8 + T cells from OT-I transgenic mice were activated with 10 ng OVA for two days or repetitively stimulated for five days to drive exhaustion. (F) Gating strategy used to define exhausted CD8 + T cells within human high-grade gliomas and surrounding tissue.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Infection, Transgenic Assay

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Representative histogram depicting CFSE dilution of mouse CD8 + T cells activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin, saxagliptin, or vehicle control for 4 days. (B) The percentage of proliferating mouse CD8 + T cells treated with sitagliptin vs. control. n=4; ** p<0.01 by paired t-test. (C) Representative zebra plots depicting Granzyme B and IFNγ production from OT-I T cells on Day 5. (D) The percentage of OT-I T cells expressing high levels of Granzyme B or IFNγ. n=4; * p<0.05 by paired t-test. (E) Representative zebra plot showing 7-AAD levels in SB28 and ovalbumin-expressing SB28 (SB28-OVA) cells co-cultured with OT-I CD8 + T cells at a 1:1 ratio. (F) The frequency of lysed wild-type SB28 or SB28-OVA cells cultured with sitagliptin- or vehicle-treated OT-I T cells. n=5; * p<0.05, ns=not significant by paired t-test.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Mouse CD8 + T cells activated with anti-CD3/CD28 plus 100IU IL-2 in the presence of 100 μM sitagliptin or vehicle control for 4 days were intracellularly stained with Ki67 to determine proliferative capacity. n=4; *** p<0.001 by paired t-test. (B) Human CD8 + T cells isolated from healthy PBMCs were expanded with anti-CD3/CD28 and 100IU IL-2 for 6 days, and CFSE dilution determined by flow cytometry. n=5; * p<0.05 by paired t-test . (C) Schematics of the killing assay. Splenic CD8 + T cells from transgenic OT-I mice were isolated and activated with 10 ng OVA for 2 days, co-cultured at 1:1 E:T with either OVA- or empty vector-expressing cancer cells for six hours and then stained with apoptotic marker 7-AAD to determine cancer cell death by flow cytometry. (D) Mouse CD8 + T cells activated with anti-CD3/CD28 plus 100 IU IL-2 in the presence of 100 μM sitagliptin or vehicle control for 4 days were stained with exhaustion marker, anti-PD-1 and anti-2B4, to determine exhaustion post treatment. n=5; * p<0.05 by paired t-test. (E) Schematics of the LCMV experiment. Naïve CD45.1 + p14 CD8 + T cells were adoptively transferred into recipient wild-type CD45.2 + mice, and then recipient mice were infected with 4 x 10 6 PFU LCMV clone 13 intravenously (i.v.) the day after. Recipient mice were treated intraperitoneally with 20 mg/kg sitagliptin daily for 5 continues doses, 2-day break, and 5 more doses for a total of 10 doses. Mice were then observed daily for indications of endpoint. (F) Splenic CD8 + T cells isolated from (E) at endpoint and stained with exhaustion marker, anti-PD-1 and anti-2B4, to determine sitagliptin induced effect in LCMV Clone 13 model. n=4; * p<0.05 by t-test. (G) Frequency of grandparents for TEX, PEX, and effector populations was determined for OT-I CD8 + T cells repetitively stimulated with 10 ng OVA for 5 days. n=7; * p<0.05, ns=not significant by paired t-test.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Control, Staining, Isolation, Flow Cytometry, Transgenic Assay, Cell Culture, Plasmid Preparation, Expressing, Marker, Infection

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin, or vehicle control for 4 days. Top differentially up-regulated pathways in sitagliptin-treated CD8 + T cells based on genes expressed >2-fold with p<0.05. n=6. Mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin, or vehicle control for 4 days and changes in cellular bioenergetics were determined with a Seahorse assay. (B) Oxygen consumption rate (OCR) and (C) Maximal respiration (top) and reserve capacity (bottom) of sitagliptin- vs. vehicle-treated CD8 + T cells. n=6; *** p<0.001, **** p<0.0001 by t-test. (D) Mouse CD8 + T cells were activated for 2 days and stained with the mitochondrial membrane potential sensitive dye TMRM at 100 nM for 30 minutes. TMRM MFI of vehicle- vs sitagliptin-treated CD8 + T cells. n=5; **p<0.01 by paired t-test. (E) Proton efflux rate (PER) and (F) extracellular acidification rate (ECAR) of CD8 + T cells from B, C. n=6. (G) CD8 + T cells activated for two days were incubated with the glucose mimetic, 2-NBDG (50 μM) for 30 min. The percentage of T cells with 2-NBDG uptake was determined by flow cytometry. n=4; ** p<0.01 by paired t-test.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Control, Staining, Membrane, Incubation, Flow Cytometry

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Volcano plot of differentially regulated genes (1799 down and 762 up) upon sitagliptin treatment for 4 days. (B) Top differentially down-regulated pathways in sitagliptin-treated CD8 + T cells based on genes expressed >2-fold with p<0.05. n=6. (C) Splenic mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin or vehicle control for 2 days and then stained with 20 nM of the mitochondrial permeable stain, Mitotracker green, for 30 minutes to quantify mitochondria mass. n=4; ns=not significant by paired t-test. (D) Splenic mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin or vehicle control overnight. Cells were collected and lysed with RIPA buffer containing phosphatase/protease inhibitors, and 20 μM of protein were loaded on an SDS-Page for protein resolution, and western blotting analysis. (E) Splenic mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin or vehicle control for 2 days, and then ATP levels were determined with CellTiter-Glo Luminescent Cell Viability Assay. n=4; ** p<0.01 by paired t-test. (F) CD8 + T cells activated for two days were incubated with the superoxide indicator, MitoSOX Deep Red, at 500 nM for 60 minutes. The percentage of T cells positive for superoxide accumulation was determined by flow cytometry. n=4; * p<0.05 by paired t-test. (G) CD8 + T cells from OT-I mice were isolated and stimulated with 10 ng OVA repetitively for 5 days to elicit exhaustion. On Day 6, cells were incubated with Mitotracker and TMRM. TMRM MFI was normalized to mitochondrial mass. n=4; * p<0.05 by paired t-test.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Control, Staining, SDS Page, Western Blot, Cell Viability Assay, Incubation, Flow Cytometry, Isolation

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Targeted metabolomics was performed with mouse CD8 + T cells activated for two days with 100 μM sitagliptin or vehicle control. Top enriched metabolic pathways were determined by >1.3-fold increase using Metaboanalyst, n=4. (B) T cell receptor (TCR) signaling-focused phosphoproteomics was performed with CD8 + T cells activated overnight. Top protein hits that were upregulated or phosphorylated >2-fold in sitagliptin- vs vehicle-treated CD8 + T cells. (C) Graphical abstract of how GAD1 feeds glutamate to the TCA cycle. (D) Representative histograms depicting CFSE dilution of CD8 + T cells expanded in the presence of sitagliptin, the GAD1 inhibitor, L-Allylglycine (100 μM), or combination treatment for 6 days. (E) Fold change in the frequency of proliferating CD8 + T cells normalized to background activation. n=4; * p<0.05, ** p<0.01, *** p<0.001 by one–way ANOVA. (F) OCR, and (G) reserve capacity of CD8 + T cells after 4 days of treatment with sitagliptin, L-Allylglycine (100 μM), or combination treatment, n=6; ** p<0.01, **** p<0.0001 by ordinary one–way ANOVA.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Control, Phospho-proteomics, Activation Assay

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Top differentially altered pathways in DPP4 high vs. DPP4 low human GBM-infiltrating CD8 + T cells. Pathways with an adjusted p-value < 0.05 were considered significantly enriched. (B) Phospho-proteomic array fluorescent signal from splenic CD8 + T cells pooled from 4 biological replicates and activated overnight with anti-CD3/CD28 plus 100 IU/mL IL-2 with 100 μM sitagliptin or vehicle control. (C) Differentially regulated pathways from (B) . (D) Targeted metabolomics was performed with mouse CD8 + T cells activated for two days with 100 μM sitagliptin or vehicle control. Metabolite quantification for GABA shunt pathway intermediates was determined. n=4; * p<0.05, ** p<0.01, **** p<0.0001, ns=not significant by paired t-test. (E) Human CD8 + T cells from healthy human donors were isolated form PBMCs and activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of sitagliptin (100 μM), the GAD1 inhibitor, L-Allylglycine (100 μM), or combination treatment for 6 days. Cells were then collected, stained with trypan blue, and quantified using an automatic cell counter. n=4; * p<0.05, *** p<0.001 by mixed effect analysis. Human CD8 + T cells from healthy human donors were isolated from PBMCs and expanded with anti-CD3/CD28 plus 100 IU/mL IL-2 for 6 days to reduce donor variability. Cells were then further activated in the presence of sitagliptin (100 μM), L-Allylglycine (200 μM), or combination treatment for four days. Seahorse T-Cell Fitness Assay was performed to determine (F) OCR and (G) reserve capacity. n=6; * p<0.05 by Friedman test.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Control, Isolation, Staining

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: Mouse CAR T-cells targeting B7-H3 were cultured with cancer cells expressing B7-H3 or empty vector at (A) 2:1 effector:target (E:T) ratio collected after 3-4 days, and co-cultured with fresh cancer cells until CAR T-cells stopped persisting in culture, or (B) at decreasing E:T ratios during the 3 rd stimulation to determine cancer cell cytotoxicity with MTS reagent after 72-hour co-culture. Heatmap demonstrating the killing capacity of either (C) sitagliptin- or (D) vehicle-treated B7-H3 CAR T-cells at reducing E:T ratio across different restimulations. (E) Normalized flux signal on day 35 post-intracranial injection n=4; * p<0.05 by t-test. (F) Diagram depicting the mechanism by which DPP-4 inhibition promotes metabolic/transcriptomic rewiring to enhance the effector function of CD8 + T cells.

Article Snippet: Mouse CD8 + T-cells were isolated from freshly harvested spleens of C57BL/6 mice using CD8a (Ly-2) MicroBeads (Miltenyi #130-114-044), following manufacturer’s instructions.

Techniques: Cell Culture, Expressing, Plasmid Preparation, Co-Culture Assay, Injection, Inhibition