T-Cell Memory for Immune Cell Function
During a primary infection, the adaptive immune system responds by identifying appropriate effector T cells, expanding and transitioning cells into a wide range of functions tailored to fight the specific immunological challenge. Once this infection has been eliminated, the majority of adaptive T cells undergo apoptosis leaving behind an array of heterogeneous memory T cells. These cells are antigen-specific T cells that remain long after an infection has been eliminated. Upon re-exposure to the same antigen the memory T cells are then quickly converted into large numbers of effector cells, thus providing a rapid response to previously seen infections.
Both vaccine and adoptive cell transfer immunotherapy protocols are influenced by T memory cell heterogeneity and it is important to be able to use a single method for identifying these functionally distinct subsets of T memory cells. Traditional methods for measuring T cell memory phenotypes and cytokine release often:
- Require separate assays for cytokine (e.g. ELISA) and marker expression analysis (e.g. traditional flow cytometry)
- Use instrumentation with low-throughput acquisition and large sample volume requirements
- Necessitate manual analysis and compilation of data from multiple sources
- Involve lengthy, time-consuming workflows; requiring steps such as protocol optimization, fixation and repetitive washes
The iQue® Human T Cell Memory application utilizes the iQue® Human T Cell Memory Kit for simultaneous, high throughput analysis of T cell memory phenotype and secreted effector proteins and cytokines. Combining the power of iQue® Advanced High-throughput Flow Cytometry with real-time data analysis using integrated iQue Forecyt® software provides a simplified solution for quantifying memory T cell populations that can be applied to immuno-oncology therapy development and investigation.
Figure 1. Illustration of iQue® Human T Cell Memory Kit assay principles.
Basic T cell phenotypes are measured by staining with CD3, CD4, and CD8 markers. T cell subsets (TN, TSCM, TCM, TTM, TEM, TEMRA, and TTE) at different stages of differentiation after activation, are measured by staining with CD45RA, CD45RO, CD27, CD62L, and CD95 markers. Secreted pro-inflammatory cytokine IFNγ and anti-inflammatory cytokine IL-10 are also quantified by Qbeads in a sandwich immune assay format in the same assay well. Live immune cells are distinguished from dead cells by staining with a fluorescent membrane integrity dye.
- Gain biological insights - Study T cell memory phenotype in physiologically relevant models
- Increase your productivity - Simultaneous measurement of cell markers and secreted cytokines in a mixed cell and beads assay format
- Streamline data analysis - Real time data analysis and novel visualization tools enable rapid generation of quantitative readouts
- Save time and precious sample - Collapse traditional workflows into one miniaturized, multiplex assay
Gain Biological Insights
Figure 2. Cytokine stimulation allows memory cells with high self renewal capacity to be maintained over 5 days.
PBMCs (100 K/well) were seeded in monoculture with ‘Core’ cytokines (IL-4, 100 ng/mL and IL-7, 10 ng/mL) and Dynabeads CD3/CD28 (25 K/well). Cells were treated with various combinations of cytokines (IL-15, 10 ng/mL; IL-6, IL-21, and IFNß, 100 ng/mL) and 10 µL samples were taken on Day 2 & 5 for analysis using the iQue Human T Cell Memory Kit.
Inclusion of IFNß (hashed bars) causes the increase in production of IL-10 and also increases the proportion of CD8+ T central memory cells by 3 fold. Combination with alternative interleukins (e.g. IL-6) has no enhancement on IL-10 production or TCM population.
Data shown is from a large panel assay designed to optimize culture conditions for bio-manufacturing of adoptive cell therapies. The heat map shows the TCM cells in all the treatments tested. Those outlined by teal box are treated with IFNß and have higher percentages of TCM cells than other treatment types.
Figure 3. High self renewal potential memory cells are not always maintained when CAR expression is induced into T-cells.
HER-2 and CD19 CAR-Ts were thawed. Control T cells from the same donor were activated over 3 days with Dynabeads CD3/CD28. Cells were labeled (100K/well in 5 replicates) with the T cell memory antibody panel to assess phenotypes. (A) Schematic showing surface marker expression changes on T cells as they develop into different T memory phenotypes. From naive (TN) to Stem Cell Memory (TSCM) to Central Memory (TCM) to Transitional Memory (TTM) to Effector Memory (TEM) to Terminal Effector (TTE) T cells through to cell death. (B) Activated T cells, (C) HER-2 CAR-T and (D) CD19 CAR-T memory phenotypes.
Increase Your Productivity
Figure 4. T cell memory development varies between donors.
PBMCs (120K/well) isolated from 3 separate donors were co-cultured with Ramos cells (3:1 E:T ratio) and activated with increasing numbers of Dynabeads CD3/CD28. 10 µL samples were analyzed at 72 hours using the iQue® Human T Cell Memory Kit.
(A) Donor 1, (B) Donor 2, (C) Donor 3, graphs showing % of CD3+ cells expressing proteins from each stage of the T cell memory development. IL-10 release is also shown. Teal bars are controls with no activator. Grey bars represent 3 ascending concentrations of Dynabeads (light to dark): 60K, 120K and 480K beads/well.
Streamline Data Analysis
Figure 5. Pre-determined gates on iQue Forecyt® enable automatic phenotyping of T cell subsets
PBMCs (120K/well) from three different donors were activated with a range of densities of Dynabeads CD3/CD28. Analysis was performed on day 3 using the iQue® Human T Cell Memory Kit. (A) Plot shows gating strategy utilized to separate T cell memory populations Central Memory (TCM) to Transitional Memory (TTM) and Effector Memory (TEM). (B) Heat map shows the proportion of T cells from each donor that are expressing the TEM phenotype. Donor 2 showed enhanced sensitivity of TEM phenotype formation at lower Dynabead densities compared to donors 1 and 3. (C) Data from (B) summarized as concentration response curves.
Save Time and Precious Sample
Figure 6. Easy to follow protocol for the analysis of T cell phenotypes and cytokine release using the iQue® Human T Cell Memory Kit.
Cell marker expression data and supernatant cytokine concentrations are acquired from a single well using a mixed cell and bead based assay. This streamlined workflow requires no additional optimization or dilutions and includes only a single wash step; minimizing time to results.
Frequently Asked Questions
Does this assay allow for intracellular cytokine detection together with secreted cytokine detection at the same time?
The iQue® Human T Cell Memory Kit is supplied with iQue QBeads® for the detection of both IFNγ and IL-10 however it only allows for the measurement of these cytokines when they are secreted into the supernatant.
The assay has been validated using a minimum seeding density of 1 x 106 cells/mL and therefore is recommended for the start of the assay. However if you anticipate a rare cell event/subpopulation then the number of cells must be increased to accommodate this to make sure the data is statistically relevant. Depending on the number of cells added it is also important to ascertain whether the cytokine levels can still be detected. If cytokine levels are too high to be measured against the linear part of the standard curve, it may be necessary to dilute the sample.
The iQue® Human T Cell Memory Kit allows for a single assay plate to be used for multiple measurements during a time course. Only a tiny volume (10 µL) of cell and supernatant sample is needed to perform analysis using the T Cell Memory Kit, meaning that once each sample has been taken (aseptically in a hood) the assay plate can be returned to the incubator ready for the next sample in your time course. Before taking a sample it is recommended to gently triturate the contents to ensure even sampling however this will not affect the cells nor will the small sample volume.