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What Our Scientist Says
Isolating T cells doesn't have to take a long time. We developed this 8-minute T cell isolation kit so you can get to your downstream experiments sooner.
Starting with human peripheral blood mononuclear cells (PBMCs), the T cell content (CD3+) of the isolated fraction is typically 96.7 ± 1.5% (mean ± SD).
Figure 2. ImmunoCult™-XF T Cell Expansion Medium Supports Faster T Cell Expansion Than Other Serum-Free and Serum-Supplemented Media
T cells were isolated from human peripheral blood samples using the EasySep™ Human T Cell Isolation Kit (Catalog #17951), stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #10970), and cultured in ImmunoCult™-XF T Cell Expansion Medium supplemented with rhIL-2. T cells were stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator on Day 0 and every 7 to 8 days for the duration of the culture. T cells were analyzed on Days 4, 7, 8, 10, 11, 14, 18, and 21 for fold expansion relative to the initial cell seeding density. Compared to all competitor media tested, ImmunoCult™-XF T Cell Expansion Medium showed significantly higher expansion of total T cells. Commercial alternatives 1 to 4 include, in no particular order, X-VIVO™ 15 (Lonza), AIM V® Medium (Life Tech), CellGro® DC Medium (CellGenix), and RPMI 1640 + serum. Each data point represents the mean fold expansion ± S.E.M. at the specified time points (p<0.05 for ImmunoCult™-XF versus all media for Days 8, 11, 14, 18, and 21, tested using two-tailed, paired t-test with unequal variance, n = 6 to 19 donors). The average fold expansion of T cells in ImmunoCult™-XF T Cell Expansion Medium were 15-fold on Day 7, 80-fold on Day 10, 450-fold on Day 14, and 4,000-fold on Day 21.
Figure 3. T Cell Expansion and Activation Ability Is Better Preserved When Leukopaks are Stored at Fridge Temperature
Using EasySep™ Human T Cell Isolation Kit (Catalog #17951), T cells were isolated from 1 leukopak fraction (Catalog # 70500) of each storage condition daily for 5 days, and 1 x 10⁶ isolated cells were cultured in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) supplemented with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog # 10970) and 10 ng/mL IL-2 for 10 days with assessment of CD25 activation marker expression on day 3 of culture. (A) Representative flow cytometry data from leukopaks stored 1 day at fridge temperature (FT), showing that both CD4+ and CD8+ cells are CD25-negative at the start of culture (Day 0), and upregulate CD25 expression by Day 3 of culture. (B) Cellular expansion and corresponding cell yield over 7 days of culture decreases in correlation with storage duration of leukopak fractions. Leukopaks that were stored for 1 - 2 days at either room temperature (RT) or FT had high expansion potential yielding 1.5 - 2 x 10⁷ cells, and this 15 - 20-fold expansion potential is maintained in T cells from leukopaks stored at FT for up to 5 days. In contrast, little or no expansion is observed in T cell cultures from leukopaks stored at RT for 3 or more days, indicating a loss of proliferative capacity. Moreover, T cells show a gradual reduction in their ability to become activated by ImmunoCult™ T Cell activator, as shown by a reduction in Day 3 CD25 expression in gated CD4+ (bottom left) or CD8+ (bottom right) cells over time, and the effect is most pronounced with storage at RT for 3 or more days. All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.
Figure 4. ImmunoCult™ Human T Cell Activators Can Be Used to Optimize Culture Conditions for High-Efficiency TRAC Knockout from Human Primary T Cells
The TRAC locus of human primary T cells was edited with an RNP-based-CRISPR-Cas9 system using multiple T cell activation reagents and dynamics then evaluated to identify a condition with the highest knockout efficiency. (A) TRAC knockout efficiency in human T cells isolated using EasySep™ Human T Cell Isolation Kit (Catalog #17951) and activated with either ImmunoCult™ Human CD3/CD28 or CD3/CD28/CD2 T Cell Activator (Catalog #10971/10970) for 2 or 3 days was assessed by binding the TCRαβ and CD3 receptors with antibodies and performing flow cytometry analysis. Each data point per condition represents an individual donor; n = 4 - 8 donors. Error bars represent standard error of the mean. (B) Genome editing (cleavage) efficiency was assessed at 48 hours post electroporation in human T cells activated with ImmunoCult™ Human CD3/CD28 T Cell Activator for 3 days using the ArciTect™ T7 Endonuclease I Kit (Catalog #76021). Mock electroporated: - RNP; RNP electroporated: + RNP. (C - D) Representative dot plots of TCRαβ and CD3 flow cytometry analysis from (C) mock electroporated and (D) RNP electroporated human T cells activated with ImmunoCult™ Human CD3/CD28 T Cell Activator for 3 days. (E) Representative dot plot of CD4 and CD8 flow cytometry analysis of human T cells activated with ImmunoCult™ Human CD3/CD28 T Cell Activator for 3 days.
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Can EasySep™ be used for either positive or negative selection?
Yes. The EasySep™ kits use either a negative selection approach by targeting and removing unwanted cells or a positive selection approach targeting desired cells. Depletion kits are also available for the removal of cells with a specific undesired marker (e.g. GlyA).
How does the separation work?
Magnetic particles are crosslinked to cells using Tetrameric Antibody Complexes (TAC). When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a separate fraction.
Which columns do I use?
The EasySep™ procedure is column-free. That's right - no columns!
How can I analyze the purity of my enriched sample?
The Product Information Sheet provided with each EasySep™ kit contains detailed staining information.
Can EasySep™ separations be automated?
Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.
Can EasySep™ be used to isolate rare cells?
Yes. We recommend a cell concentration of 2x108 cells/mL and a minimum working volume of 100 µL. Samples containing 2x107 cells or fewer should be suspended in 100 µL of buffer.
Are the EasySep™ magnetic particles FACS-compatible?
Yes, the EasySep™ particles are flow cytometry-compatible, as they are very uniform in size and about 5000X smaller than other commercially available magnetic beads used with column-free systems.
Can the EasySep™ magnetic particles be removed after enrichment?
No, but due to the small size of these particles, they will not interfere with downstream applications.
Can I alter the separation time in the magnet?
Yes; however, this may impact the kit's performance. The provided EasySep™ protocols have already been optimized to balance purity, recovery and time spent on the isolation.
For positive selection, can I perform more than 3 separations to increase purity?
Yes, the purity of targeted cells will increase with additional rounds of separations; however, cell recovery will decrease.
How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?
Hundreds of publications have used cells selected with EasySep™ positive selection kits for functional studies. Our in-house experiments also confirm that selected cells are not functionally altered by the EasySep™ magnetic particles.
If particle binding is a key concern, we offer two options for negative selection. The EasySep™ negative selection kits can isolate untouched cells with comparable purities, while RosetteSep™ can isolate untouched cells directly from whole blood without using particles or magnets.
IL-6 drives T cell death to participate in lymphopenia in COVID-19. X. Zhou et al. International immunopharmacology 2022 oct
Abstract
Lymphopenia is a common observation in patients with COVID-19. To explore the cause of T cell lymphopenia in the disease, laboratory results of 64 hospitalized COVID-19 patients were retrospectively analyzed and six patients were randomly selected to trace their changes of T lymphocytes and plasma concentration of IL-6 for the course of disease. Results confirmed that the T-cell lymphopenia, especially CD4+ T cell reduction in COVID-19 patients, was a reliable indicator of severity and hospitalization in infected patients. And CD4+ T cell count below 200 cells/$\mu$L predicts critical illness in COVID-19 patients. In vitro assay supported that exposure to key contributors (IL-1$\beta$, IL-6, TNF-$\alpha$ and IFN-$\gamma$) of COVID-19 cytokine storm caused substantial death of activated T cells. Among these contributors, IL-6 level was found to probably reversely correlate with T cell counts in patients. And IL-6 alone was potent to induce T cell reduction by gasderminE-mediated pyroptosis, inferring IL-6 took a part in affecting the function and status of T cells in COVID-19 patients. Intervention of IL-6 mediated T cell pryprotosis may effectively delay disease progression, maintain normal immune status at an early stage of infection.
Expanding anti-CD38 immunotherapy for lymphoid malignancies. X. Wang et al. Journal of experimental & clinical cancer research : CR 2022 jun
Abstract
BACKGROUND Lymphoid neoplasms, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and NK/T cell neoplasms, are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies, like daratumumab, have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials. METHODS We generated chimeric antigen receptor (CAR) T cells targeting CD38 and tested its cytotoxicity against multiple CD38high and CD38low lymphoid cancer cells. We evaluated the synergistic effects of all-trans retinoic acid (ATRA) and CAR T cells or daratumumab against cancer cells and xenograft tumors. RESULTS CD38-CAR T cells dramatically inhibited the growth of CD38high MM, mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia (WM), T-cell acute lymphoblastic leukemia (T-ALL), and NK/T-cell lymphoma (NKTCL) in vitro and in mouse xenografts. ATRA elevated CD38 expression in multiple CD38low cancer cells and enhanced the anti-tumor activity of daratumumab and CD38-CAR T cells in xenograft tumors. CONCLUSIONS These findings may expand anti-CD38 immunotherapy to a broad spectrum of lymphoid malignancies and call for the incorporation of ATRA into daratumumab or other anti-CD38 immunological agents for cancer therapy.
Indomethacin-induced oxidative stress enhances death receptor 5 signaling and sensitizes tumor cells to adoptive T-cell therapy. N. S. Aboelella et al. Journal for immunotherapy of cancer 2022 jul
Abstract
BACKGROUND Adoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However, even for the best-studied and clinically validated CD19-targeted chimeric antigen receptor (CAR) T-cell therapy, many patients face the challenge of lack of response or occurrence of relapse. There is increasing need to improve the efficacy of ACT so that durable, curative outcomes can be achieved in a broad patient population. METHODS Here, we investigated the impact of indomethacin (indo), a non-steroidal anti-inflammatory drug (NSAID), on the efficacy of ACT in multiple preclinical models. Mice with established B-cell lymphoma received various combinations of preconditioning chemotherapy, infusion of suboptimal dose of tumor-reactive T cells, and indo administration. Donor T cells used in the ACT models included CD4+ T cells expressing a tumor-specific T cell receptor (TCR) and T cells engineered to express CD19CAR. Mice were monitored for tumor growth and survival. The effects of indo on donor T cell phenotype and function were evaluated. The molecular mechanisms by which indo may influence the outcome of ACT were investigated. RESULTS ACT coupled with indo administration led to improved tumor growth control and prolonged mouse survival. Indo did not affect the activation status and tumor infiltration of the donor T cells. Moreover, the beneficial effect of indo in ACT did not rely on its inhibitory effect on the immunosuppressive cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis. Instead, indo-induced oxidative stress boosted the expression of death receptor 5 (DR5) in tumor cells, rendering them susceptible to donor T cells expressing TNF-related apoptosis-inducing ligand (TRAIL). Furthermore, the ACT-potentiating effect of indo was diminished against DR5-deficient tumors, but was amplified by donor T cells engineered to overexpress TRAIL. CONCLUSION Our results demonstrate that the pro-oxidative property of indo can be exploited to enhance death receptor signaling in cancer cells, providing rationale for combining indo with genetically modified T cells to intensify tumor cell killing through the TRAIL-DR5 axis. These findings implicate indo administration, and potentially similar use of other NSAIDs, as a readily applicable and cost-effective approach to augment the efficacy of ACT.
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