Q&A Report: Assessing Antigen-Specific T Cell Functionality With Dendritic Cell/CD8⁺ T Cell Co-Culture

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 1×10⁶ 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 and 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 Fig. 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.

We have successfully expanded CMV-reactive T cells by starting with whole PBMCs. However, you don’t get the same yield as when you use purified T cells and DCs. The DC/T cell co-culture is especially beneficial when using naive T cells, as DCs are best suited for activating naive T cells.

We haven’t tracked activation marker expression throughout the culture period. You may notice upregulation of the CD154 and CD69 activation markers in the first few days and they may be upregulated again in response to various stimuli or re-exposure to antigens. Some ways of minimizing non-specific CD4⁺ T cell activation are to optimize cytokine concentrations and to omit serum (we didn’t use any) to avoid basal levels of activation during the co-culture.

Tissue-derived DCs can be a very heterogeneous mixture, so not all cells will respond equally to maturation signals. Consequently, the combination of signals the DCs generate will differentially impact T cell priming and differentiation. We haven’t tested this in-house yet.

In our experience, both positively and negatively selected monocytes can be differentiated into DCs, but the yield is much higher with the negative selection approach. Either approach will generate mo-DCs that are capable of presenting antigens, but keep in mind that the antigen presentation/phagocytosis could be different between the positive and negative monocyte-enrichment approaches. We have not directly measured this in-house yet.

For T cell priming, DCs would be the best choice as they express cytokines and co-stimulatory molecules that can potently activate naive or memory T cells. However, for functional assays such as CD8-mediated killing assays, or antigen-induced cytokine production, non-DC class I-expressing cells can be used, provided they express the appropriate HLA class I molecules.

Circulating DCs are very rare and a bit more finicky to culture. The kind of DC subset will determine the maturation signals you will need. You may also have to optimize DC to T cell ratios.

Since we haven’t done a head-to-head comparison of the two cell types, I can’t comment on whether they are comparable or better.

We haven’t tested this in-house yet. You can refer to the literature to see what’s currently being done and how this can be implemented in your workflow.

With a few optimizations, the system can be used in mice. According to the literature, bone marrow (BM) is commonly used as a source of myeloid cells. T cells isolated from tissues such as the spleen typically have more cells than blood. Overall the idea is similar, where myeloid cells can be differentiated into bone marrow-derived dendritic cells (BMDCs) and CD8⁺ T cells can be isolated from the spleen. However, factors such as cytokine concentration, combinations, culture duration, DC maturation requirements, etc. may all be different than their human counterparts and should be optimized for mouse applications.

Mitomycin C is used to prevent cell division and expansion. Monocytes will not expand in these culture systems, so mitomycin C is not needed unless there are contaminants after mo-DC differentiation, such as T or NK cells.

Yes, mo-DCs will be quite adherent after the 5-day mo-DC differentiation protocol. They may require reagents such as EDTA (recommended) or trypsin to detach them.

A very preliminary experiment performed in-house showed that repeatedly restimulating human T cells with an ImmunoCult™ T cell activator after a primary stimulation resulted in an exhausted phenotype. For this, T cells were stimulated every 2-3 days for a total of 14 days of culture in ImmunoCult™-XF medium. The activator doses were administered at a concentration of 25μL/mL. The exhaustion level can be assessed by evaluating the expression of surface markers PD-1, TIM-3, and LAG3, as well as the transcription factor TOX. Additionally, the functionality of the cells can be determined by measuring their production of cytokines such as IL-2, IFN-γ, TNF-α, and Granzyme B upon restimulation and reduced proliferation capacity. v

Mouse CD8⁺  T cells can be used in co-culture with bone marrow-derived dendritic cells (BMDCs) to study antigen-specific CD8⁺ T cell responses. The BMDCs must express the same MHC class I molecules as the CD8⁺ T cells.

Other commonly used peptide pools (except CMV) will include peptides derived from EBV or influenza. If a tetramer is not available to track expansion, other readouts such as CD137 or interferon-gamma production can be used to monitor and detect antigen-specific CD8⁺ T cells.

Human AB serum may contain fewer components that can induce non-specific T cell activation compared to FBS. However, there is lot-to-lot variability among vendors and donors of AB serum, and some level of quality control and re-optimization may be required for your assays.

Prolonged exposure to trypsin may affect the level of MHC and co-stimulatory proteins on the surface of the mo-DCs. You can try to incorporate a recovery step, which may allow the cells to re-express the proteins needed to promote the priming of T cells. This would require some optimization.

It is possible, depending on your research question. Immature mo-DCs will have reduced expression of MHC I/II, ligands for co-stimulation, and IL-12 production compared to matured mo-DCs. Hence, the level of T cell priming may be weaker.

It’s possible to promote further expansion of T cells with aCD3/aCD28 reagents. However, this will expand all T cells, not just antigen-specific T cells. This may or may not benefit downstream assays.                                                                                                                                                                             

We do not have any in-house experience with the delivery of RNA to mo-DCs. The maturation supplement may or may not be necessary and It will depend on whether the RNA or RNA delivery mechanism can promote mo-DC maturation on its own.

If you have mo-DCs and CD8⁺ T cells from the same donor, it is possible to use this technique to study antigen-specific CD8⁺ T cell responses in other species. However, you may need to rely on readouts such as IFN-gamma production or molecular methods to detect T cell activation (depending on what commercially available antibodies/ tetramers are available).