Cancer Immunology & Immune Cell Engineering

01

Immune Killing

Immune Cell Killing Assays

I design and execute T-cell mediated killing, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and optimize effector-to-target ratios to profile immune potency and mechanistic dependencies. Assays employ primary human T-cells, NK cells, and macrophages in co-culture with fluorescently-labeled or viability-dye-stained target cells, with quantification by flow cytometry or luciferase-based readouts.

RT-qPCR — Immune Cell Marker Expression

RT-qPCR quantification confirms cell-type identity and treatment response at the transcriptional level. Expression is normalized to housekeeping genes (GAPDH, ACTB) and presented as fold change relative to undifferentiated or untreated controls. Error bars represent SEM from n=3 biological replicates.

RT-qPCR: Immune Checkpoint & Cytokine Profiling

Fold change in T-cell markers and cytokines across co-culture conditions vs unstimulated PBMCs

ΔΔCt method. n=3 donors. Stimulation: anti-CD3/CD28 ± tumor co-culture (48h). Housekeeping: GAPDH, B2M.

RT-qPCR Expression Heatmap

Relative gene expression across conditions (log₂ fold change)

Color intensity: red = upregulated, blue = downregulated relative to control. Values are log₂(fold change).

T-Cell Killing Dose-Response

Fig. 1A — T-Cell Mediated Cytotoxicity

Dose-dependent lysis of target cells by anti-PD-L1-enhanced T-cells across effector-to-target ratios

Primary human T-cells co-cultured 4h with labeled target cells. Anti-PD-L1 shows checkpoint-dependent enhanced killing. Specific lysis calculated as (experimental release − spontaneous release) / (maximum release − spontaneous release) × 100%.

ADCC Activity Profile

Fig. 1B — Antibody-Dependent Cellular Cytotoxicity

Comparative ADCC activity of therapeutic antibodies at E:T 10:1

Freshly isolated PBMCs (effector) co-cultured with antibody-opsonized target cells. Trastuzumab and Rituximab show superior ADCC compared to Cetuximab. Isotype control minimal background.

CDC Kinetics

Fig. 1C — Complement-Dependent Cytotoxicity Over Time

Kinetic profile of complement-mediated target cell lysis

Target cells opsonized with anti-target antibody ± human complement serum. Maximal lysis achieved by 2h. Antibody or complement alone show minimal activity.

02

Checkpoints

Checkpoint Biology & Immune Modulation

I characterize the expression and functional role of inhibitory checkpoint receptors (PD-1, CTLA-4, LAG-3, TIM-3, TIGIT) on tumor-infiltrating and circulating T-cells. Checkpoint blockade assays measure rescue of exhausted T-cell function, IFN-γ secretion, and proliferation using anti-checkpoint monoclonal antibodies and ligand-blocking strategies.

Checkpoint Expression Profile

Fig. 2A — Checkpoint Receptor Expression

Mean fluorescence intensity (MFI) fold-change on tumor-infiltrating T-cells vs peripheral blood T-cells

Multicolor flow cytometry. TILs show elevated checkpoint expression consistent with T-cell exhaustion. PB = peripheral blood T-cells; TIL = tumor-infiltrating lymphocytes.

Blockade Functional Assay

Fig. 2B — Checkpoint Blockade Rescues T-Cell Function

IFN-γ secretion by exhausted T-cells ± anti-checkpoint antibodies

Exhausted T-cells (PD-1 high) stimulated with anti-CD3/CD28 ± anti-PD-1, anti-CTLA-4, or combination. Dual blockade shows superior functional rescue. Quantified by multiplex bead assay (Luminex).

03

Cytokines

Cytokine Profiling & Biomarker Discovery

I employ multiplex immunoassays including Luminex, MSD V-PLEX, and AlphaLISA to quantify immune-relevant cytokines (IL-2, IL-6, IL-10, IFN-γ, TNF-α, IL-17A, Granzyme B) from T-cell co-cultures and immune-oncology screens. Dose-response profiling identifies optimal pathway modulation and therapeutic windows.

Cytokine Panel Heatmap

Fig. 3A — Comprehensive Cytokine Panel

Multiplex quantification of immune activation markers across conditions

Supernatants from T-cell co-cultures collected at 48h. Unstim = resting; Stim = anti-CD3/CD28; Stim+Drug = stimulated + checkpoint inhibitor. Values normalized to unstimulated baseline.

Dose-Response Cytokine

Fig. 3B — Dose-Dependent Cytokine Secretion

IL-2 and IFN-γ titration profiles across drug concentrations

T-cells stimulated with checkpoint inhibitor at increasing concentrations (0.01–100 nM). Both cytokines show dose-dependent increase with plateau at higher concentrations. EC50 determination informs optimal dosing.

04

CAR-T

CAR-T & Engineered Immune Cells

I characterize CAR-T cell transduction efficiency, expansion kinetics, phenotype stability, and in vitro potency. Assays measure transduction rates by flow cytometry (CAR expression via tag-specific antibodies), expansion capacity over 14–21 days, exhaustion markers, and target-cell killing across CAR-T designs and target lineages.

CAR-T Expansion Kinetics

Fig. 4A — CAR-T Expansion Over Culture Period

Fold-expansion of transduced vs untransduced T-cells over 14 days in culture

CAR-T cells show enhanced proliferation kinetics and reach higher peak expansion (~80-fold by day 12). Untransduced T-cells plateau earlier (~20-fold). Both cultured with IL-2 supplementation and anti-CD3/CD28 stimulation.

CAR-T Potency Assay

Fig. 4B — CAR-T-Mediated Target Cell Killing

Specific lysis of CD19+ and CD19− target cells by CAR-T vs untransduced T-cells

CAR-T (anti-CD19) co-cultured with Raji, Nalm-6 (CD19+), or K562 (CD19−) target cells at E:T 5:1. CAR-T shows antigen-specific killing; minimal off-target activity against K562. Untransduced T-cells show background killing.

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Methods

Detailed Methodology & Techniques

Comprehensive descriptions of key experimental techniques, assay platforms, and analytical methods referenced throughout this page.

ADCC Assays

I develop and optimize ADCC assays using NK effector cells and PBMC-derived effectors against antibody-opsonized tumor target cells. Readouts include LDH release, flow cytometric viability (Annexin V/7-AAD), and real-time impedance-based killing on the xCELLigence RTCA platform. These assays benchmark therapeutic antibody potency, Fc engineering variants, and biosimilar comparisons.

Fig. — ADCC Dose-Response by E:T Ratio

NK cell–mediated killing of HER2+ target cells at varying effector:target ratios

LDH release assay, 4 hr co-culture. n=3 replicates per condition.

CDC Assays

I establish complement-dependent cytotoxicity (CDC) assays using human serum complement against antibody-opsonized tumor cells. CDC measures the ability of therapeutic antibodies to activate the classical complement pathway (C1q binding → MAC formation). Readouts include propidium iodide uptake by flow cytometry and CellTiter-Glo viability. These assays characterize IgG1 vs IgG2 subclass activity and evaluate Fc mutations that enhance or ablate complement fixation.

Fig. — CDC Potency: Antibody Subclass Comparison

Complement-mediated lysis of CD20+ Raji cells at saturating antibody concentration

20% pooled human serum. 2 hr incubation. PI uptake by flow cytometry. n=4.

NK Cell Biology

I isolate and expand NK cells from PBMCs using IL-2/IL-15 cytokine cocktails for use in ADCC, direct cytotoxicity, and serial killing assays. NK phenotyping includes CD56, CD16, NKG2D, KIR, NKp46 by multicolor flow cytometry. Functional readouts include degranulation (CD107a surface mobilization), intracellular IFN-γ/TNF staining, and real-time killing kinetics via impedance or IncuCyte imaging.

Fig. — NK Cell Activation Markers

Functional marker expression in resting, IL-2 activated, and target-engaged NK cells

Intracellular staining + CD107a surface assay. Flow cytometry. n=3 donors.

Immune Co-Culture Systems

I establish immune–tumor co-culture assays combining patient-derived PBMCs, purified T-cell subsets, or NK cells with tumor target cells at defined E:T ratios. These enable evaluation of checkpoint blockade efficacy, bispecific antibody potency, and CAR-T cytotoxicity. Readouts include real-time impedance (xCELLigence), endpoint viability (CTG/LDH), and multiplexed cytokine profiling (MSD V-PLEX).

Cancer Immunology & Immune Cell Assay Platform

An integrated assay framework for characterizing tumor–immune interactions across T-cell killing, ADCC/CDC, checkpoint biology, CAR-T, NK cell function, and multiplex cytokine profiling.

Effector Cells

Primary Human T-Cells

CD3⁺/CD8⁺ cytotoxic T lymphocytes. Anti-PD-L1 enhanced killing. Dose-response across E:T ratios (1:1 → 50:1).

Effector Cells

NK Cells

Freshly isolated or expanded NK cells. CD16-dependent ADCC. K562 and primary tumor target killing.

Checkpoint

Exhaustion Profiling

PD-1, CTLA-4, LAG-3, TIM-3, TIGIT MFI on TILs vs. PBMCs. Blockade rescue of IFN-γ secretion and proliferation.

Engineered

CAR-T & CAR-NK

Lentiviral CAR construct transduction. Antigen-specific killing, cytokine release, and exhaustion kinetics.

CENTRAL ASSAY

Immune–Tumor
Co-Culture

Effector + Target
4–72 h incubation

4 h

Acute killing

72 h

Extended co-culture

E:T scan

Ratio optimization

Multi-Ab

Antibody profiling

Target Cells

Tumor Cell Lines

Fluorescently labeled or viability-dye stained. Surface antigen expression validated by flow cytometry.

Readout

Flow Cytometry

Multicolor panels: CD3, CD4, CD8, CD56, PD-1, PD-L1, Ki-67. Live/dead discrimination. Intracellular cytokines.

Readout

Cytokine Quantification

MSD V-PLEX 10-plex: IFN-γ, TNF-α, IL-2, IL-6, IL-10, Granzyme B. Supernatant & intracellular staining.

Readout

Killing Quantification

Luciferase release, LDH, Annexin V, PI, CellTiter-Glo. Real-time impedance (xCELLigence) for kinetics.

Functional Assay Modalities

T-CELL KILLING

CTL Cytotoxicity

Anti-PD-L1 enhanced. Dose-response across E:T ratios. Specific lysis calculation.

ADCC

Antibody-Dependent

Trastuzumab, Rituximab, Cetuximab profiling. PBMC effectors at 10:1 E:T.

CDC

Complement-Dependent

Human complement serum. Time-course kinetics. Maximal lysis at 2 hours.

CHECKPOINT BLOCKADE

Exhaustion Rescue

Anti-PD-1, anti-CTLA-4, anti-LAG-3 monoclonal antibodies. Functional IFN-γ rescue.

Fig. — Real-Time Killing Kinetics (xCELLigence)

Impedance-based cell index tracking tumor cell viability during immune co-culture

xCELLigence RTCA. E:T = 5:1 (PBMC), 1:1 (CAR-T). n=4 wells per condition.

Cancer Immunology & Immune Cell Engineering

Immune Cell Killing Assays

Checkpoint Biology & Immune Modulation

Cytokine Profiling & Biomarker Discovery

CAR-T & Engineered Immune Cells

Detailed Methodology & Techniques

Workflow