The protocol described in our technical bulletin is most optimal. The DC maturation is done in ImmunoCult™ Dendritic Cell Medium and the co-culture is performed in ImmunoCult™-XF T Cell Expansion Medium.

We used ImmunoCult™-XF T Cell Expansion Medium. We haven't tested other media in this setup.

We provide this information in the ImmunoCult™ Dendritic Cell Kit. The cells are seeded at 1x10⁶ cells/mL and the medium is replaced on Day 3 without disturbing the cells too much. Then, you add the maturation supplement and peptides on Day 5. Minimal manipulation and handling is best for these cells. The quality of the monocytes that you start with will also affect final yields (ie. fresh vs. frozen sources, the viability of starting cells, etc). It’s expected that you will not recover the same number of cells you started with after the differentiation and maturation process.

In this case, you have several options. We recommend checking the viability and phenotype of the T cells and mature DCs for typical markers. You can also check IL-12 secretion by the DCs. We also recommend using a positive control such as CMV peptide pools (expands memory T cells) as most donors will have circulating memory T cells to CMV. Another option for a positive control is to use CEF class I peptide pools, as this is composed of CMV, EBV, and influenza peptides, and most donors will have memory T cells against these peptides.

Yes, but with a few modifications. After enriching the antigen-specific T cells for one round, try using fewer DCs and exploring how changing the cytokine concentration or combination might work for you. Some things to consider are T cell exhaustion and phenotype changes that accompany multiple rounds of expansion.

No, DCs don't tend to proliferate, as these are terminally differentiated cells. If you are starting with monocytes and differentiating them to DCs, the final yield would depend on the donor, culture method, and sample quality (ie. fresh vs. frozen sources, the viability of starting cells, etc). You should expect the yield of mo-DCs at the end of differentiation and maturation to be lower than the number of monocytes that were seeded on Day 0 of the DC differentiation workflow.

This depends on the cell type you start with. Blood-derived DCs such as the cDC1s and cDC2s would respond to different stimuli. Some researchers use TLR9 or TLR3 agonists based on the subset of DC they are working with. There are several diverse approaches and we encourage customers to try various options.

You should see a downregulation of CD14 by Day 5. They will be CD83- and have much lower levels of co-stimulatory molecules and MHC levels compared to mature DCs. Please see Figure 2 on this scientific poster for more details.

When using Pan-T cells isolated using a CD3 positive selection kit, there may be some basal levels of stimulation of T cells that may be caused by the anti-CD3 antibody in the kit binding to the cells. Other markers such as CD4 may be expressed on non-T cells so you may see some contaminating cells such as monocytes. CD8 can also be expressed on NK/T and NK cells that also respond to IL-2 or IL-15 and can compete with T cells. For these reasons, it is better to opt for negative selection as it will better deplete non-T cells.

Our mo-DC approach works reasonably well. Please refer to this technical bulletin for more information. In terms of the final yield of antigen-specific CD8⁺ T cells, adding IL-21 was beneficial. An additional suggestion is to isolate naive CD8⁺ T cells using a negative selection kit and use them in co-culture in place of pan CD8⁺ T cells. This increases the frequency of the naive antigen-specific CD8⁺ T cells present in the culture.

Overall, you can use a similar system for CD8⁺ and CD4⁺ T cells. However, optimal cytokine concentrations may be different from CD8⁺ T cells, as CD4⁺ T cells have different sensitivities to cytokines and will respond differently to them. Additionally, the CD4⁺ T cells respond to antigens complexed with MHC II, so the source of antigens and antigen-processing steps are distinct from CD8⁺ T cells. CD4⁺ T cells can also produce higher levels of IL-2 compared to CD8⁺ T cells.

Allogeneic co-cultures are quite different from the antigen-specific T cell co-culture system. The frequency of alloantigen T cells is typically higher than observed for antigen-specific T cells. Hence, you can use lower numbers of DCs and different concentrations or combinations of cytokines. You can expect some donor variability, as not all donors respond to the same alloantigens to the same extent. The most common challenge is the observation that many non-alloantigen CD4⁺ T cells will become activated and expanded, a phenomenon called "bystander activation." Titration experiments to find the most optimal concentration of cytokines (you may not need any at all) are required to try and minimize non-antigen-driven T-cell activation.

Most publications recommend cytokine concentrations in ng/mL, and do not account for differences in biological activity. The specific activity of the cytokine is more important and can vary between lot numbers and vendors. It may be best to stick to the same lot number or vendor as much as possible to reduce variability or test different amounts when using a new lot number to see what works best.

We add IL-21 on Day 0 of the co-culture. We used a final concentration of 30 ng/mL, but this needs to be optimized depending on your model system.

There are many aAPC systems that express/present a variety of molecules. Ultimately, to recapitulate DC's capacity to induce T cell priming, at least 3 signals must be present: a source of cognate peptide/MHC class I suitable for the donor's HLA type, a source of costimulation (eg. ligands for CD28), and inflammatory cytokines (such as IL-12). Additional cytokines such as IL-21 priming may still be beneficial. You can explore other combinations such as IL-15 or IL-2 as well. Anytime you change the antigen in your system, some level of optimization may be required.