Investigating T lymphocyte populations purity and viability
Setting up DC-T cell co-cutlers is generally challenging especially when it involves initial conditioning of DCs followed by setting up co-culture with T cells. In order to verify separation and isolation methods to obtain of T cells from buffy coats, purity of isolated T cells was assessed using CD3 as a general marker for all T cells. Furthermore, CD4 marker was used to confirm presence of both Th from T cytotoxic (Tc) population and CD45RA was used to differentiate between the CD45RA + naïve T cell and the CD45RA- memory T cell populations. Data obtained (Fig. 1) confirms the presence of CD3 T cell population where both CD4 + Th and CD4- (CD8+) Tc subtypes of T cells can be differentiated as well as natural killer cells which lack the expression for both markers. The expression of CD45RA show the naïve T cell population while the lack of the expression shows the memory T cells population. This confirms the separation and isolation of T cells from buffy coats and the presence of the target populations to be used in further isolation of the naïve T cell population for DC-T cell co-culture experiments as well as our allergy model experiments.
Investigating isolated naïve T lymphocyte purity
To obtain naïve T cells, further isolation steps are used on the pan T cell populations isolated from buffy coat PBMCs. This isolation focuses on depletion of CD45RO + memory cells using microbeads and the remainder population of lymphocytes would be naïve T cells of both CD4 + and CD8 + subtypes. After the isolation process, T cells were analyzed using the previously mentioned markers to assess presence of naïve CD3 + population of both CD4 + Th and CD4- (CD8+) Tc and the absence of CD45RA- memory T cells. Data in (Fig. 2) confirm presence of CD3 + population of T cells expressing CD4 + or CD4- (CD8+). Data also confirms the absence of the previously present population of CD45RA- memory cells seen in (Fig. 1) indicating the purity of the naïve cells obtained. Both CD4 + and CD8 + subtypes are present in the CD3 naïve T cell population.
Viability, stability, and potency of liquid nitrogen preserved T-cells for homologous DC-T-cell in vitro co-cultures
To be able to co-culture human primary cells, several hurdles must be overcome and most importantly is viability and stability of cells after freeze-thaw cycle. This is because the need to use donor homologous cells for co-cultures. Direct cell preserving in liquid nitrogen will reduced their viability, stability, and potency. For that optimization for preservation of donor isolated T cells for the duration of DC generation and condition treatment (up to 7 days) was addressed. The process used to preserve T cells until co-culture is illustrated in Fig. 3. Briefly, both CD14 + monocytes and T cells were isolated from the same donor where monocytes are incubated for differentiation towards DCs, and T cells (naïve or pan) were preserved using liquid nitrogen. T cells were thawed and assessed for cell viability, stability, and potency through the interaction with homologous DCs using proliferation as an indicator of cell interaction. Viability was assessed after thawing from liquid nitrogen and after 6 days of co-culture with LPS stimulated DC. T cell viability was done in both trypan blue automated counter and in flow cytometry Live/Dead assay (see materials and methods). Data in (Fig. 4) show naïve and pan T cell viability using both trypan blue automated counter and live/dead flow cytometry assay. After 7 days of liquid nitrogen preservation pan T cells had a viability of 98.4% while naïve T cells had a viability of 93.2% of live cells. Comparing this to the after-isolation viability for both T cell groups show no significant reduction in number of live cells. Trypan blue stain using countess automated counter showed same viability for both groups of T cells confirming the results. This indicates that liquid nitrogen preservation methods used are not affecting T cell viability.
After 6 days co-culture, T cells showed a viability of > 99% using Live/dead assay after DC: T cell co-culture with LPS stimulated DC (Fig. 5). This confirms the stability and the viability of T cells isolated for both naïve and pan populations. We also looked at the activation marker CD25 to understand if cells have been pre-activated before co-culture rendering them unable to interact with DCs which would provide false results. Data in (Fig. 6) show population of both naïve and pan T cells isolated where CD25 an activation marker for T cells is not expressed confirming steady state of the cells obtained after thawing from liquid nitrogen.
We have then looked T cell proliferation after co-cultures with LPS activated DCs or not to confirm T cells potency in which preserved T cells should still be able to interact with DCs and proliferate. Naïve T cells lack the memory subtype and are slower to proliferate, for that they were re-stimulated with anti-CD3 and anti-CD28 while pan T cells were not re-stimulated. Proliferation graph (Fig. 7) show that isolated T cells for both naïve and pan were able to proliferate upon co-culture with mature DCs confirming their quality and potency after liquid nitrogen thawing.
This proves that methods used for the isolation of both pan and naïve T cells produced good quality cells and the preservation methods for these cells can be utilized to further move towards homologous DC-T cell co-culture experiments.