PHENOTYPE AND IMMUNOMODULATORY PROPERTIES OF PαS MSCs
3.1 Chapter Rationale and Aims
3.2.10 Optimising the ‘Splenocyte reaction’
Previous mouse literature has identified differences in the biology of MSCs isolated from different mouse strains, although their investigations were limited to the growth and differentiation of these cells (Cunha et al., 2013, Peister et al., 2004, Phinney et al., 1999). One study looked at the immunosuppressive properties of adipose-‐derived MSC conditioned media from C57BL/6 and Balb/c mice and identified Balb/c mice to be more suppressive (Hashemi et al., 2013). However, no study has performed a comparative analysis of the immunosuppressive mechanism of Balb/c and C57BL/6 strains using a purified population of BM-‐derived MSCs.
We hypothesised that C57BL/6 MSCs may indirectly suppress lymphocyte proliferation by acting at the antigen presentation stage or on another cell (e.g. monocyte/macrophage). Our current T cell proliferation assay bypassed the antigen presentation stage via the use of antibodies or beads that bind directly to the TCR. Additionally, no supporting cells (except B cells) were present in the mixture. To test this hypothesis, I developed a new assay that used ‘bulk’ splenocytes instead of purified T and B cells (termed the ‘Splenocyte reaction’). Mouse spleen contains a variety of cell types, including T cells, B cells, dendritic cells, monocytes/macrophages and stromal cells (Hey and O'Neill, 2012). I then tested the effect of various stimulatory reagents in the splenocyte reaction to achieve a strong and reproducible proliferative effect on the T cell population.
3.2.10.1 Concanavalin A Stimulation
The plant lectin Concanavalin A (ConA) is used to trigger T cell proliferation by binding directly to the TCR (Palacios, 1982). A range of ConA concentrations (1µg/ml to 100µg/ml) was used in splenocyte cultures and incubated for 72 hours. Cultures were stained with the membrane dye cell trace violet (CTV) to assess proliferation and total numbers of CD4 and CD8 T lymphocytes was then quantified using flow cytometry. Surprisingly, all ConA concentrations tested failed to induce CD4 T cell proliferation in this assay (Figure 3.10A). Some cell division was seen with 5µg/ml ConA in the CD8 T cell samples (Figure 3.10B), but the majority of cells remained undivided. Higher doses of ConA (>20µg/ml) could not be tested on CD8 T cell samples due to cell death at higher concentrations.
Figure 3.10 | Stimulation of splenocyte cultures with ConA. Proliferation of CD4 (A) and CD8 T cells (B) in splenocyte cultures after incubation with different concentrations of ConA. Representative fluorescence histograms of two independent repeats shown.
3.2.10.2 CD3/CD28 Dynabeads® Stimulation
CD3/CD28 Dynabeads® bind directly to the TCR and CD28 to provide both the primary and secondary signals required for T cell proliferation. A range of concentrations was used to induce proliferation in our splenocyte cultures (Table 3.2). Low doses of Dynabeads® (<15,000 beads per well) failed to elicit a proliferative response in both the CD4 and CD8 populations (Figure 3.11A and B). Medium doses of Dynabeads® (100,000 to 300,000 beads per well) were able to induce a strong CD8 response (≈80% proliferated) but less than half the CD4 population responded. High doses of beads (>400,000 beads per well) were required to generate a strong response in both lymphocyte populations (Figure 3.11C and D). As such, Dynabeads® stimulation was, in our hands, a cost-‐ineffective method of inducing T cell proliferation in the splenocyte reaction and alternative approaches involving TCR-‐ specific peptide sequences were tested.
Table 3.2 | Percentage of T cell proliferation after Dynabeads® stimulation.
Number of Beads/well Percentage of CD4proliferated (% ) + T Cells that Percentage of CD8proliferated (% ) + T Cells that
0 0.18 0.67 1875 0.69 1.00 3750 0.67 1.81 7500 0.92 1.79 15,000 0.54 1.83 30,000 2.55 35.4 60,000 11.9 77.5 100,000 24.2 51.5 200,000 39.2 73.2 300,000 49.9 81.1 400,000 54.4 85.1 500,000 60.3 89.5
Figure 3.11 | Stimulation of splenocyte cultures with Dynabeads®. Proliferation of CD4 (A and C) and CD8 (B and D) T cell populations with low (upper row) and high (lower row) numbers of Dynabeads® per well. Cultures were maintained for 72 hours before analysis. N=1 biological repeat per condition.
3.2.10.3 Ovalbumin peptide stimulation
CD8 T cells from OT-‐1 transgenic mice have a TCR that is specific for the ovalbumin (OVA) peptide sequence SIINFEKL when presented in a MHC class I context (Wright et al., 2005, Clarke et al., 2000). The addition of OVA peptide to OT-‐1 splenocyte cultures causes expansion of antigen-‐specific CD8 T cells in a physiological manner. Unlike ConA and CD3/CD28 Dynabeads®, which can bind directly to T cells to cause activation, OVA peptide needs to be processed and presented alongside a co-‐stimulatory signal to cause activation and proliferation of CD8 lymphocytes. Additionally, stimulating OT-‐1 splenocytes with OVA peptide is a good in vitro correlate of the OVA-‐Bil mouse model (Chapter 6) in which adoptive transfer of OT-‐1 cells causes their migration to the liver (where OVA is expressed) and induction of inflammatory liver injury (Buxbaum et al., 2006).
The dose of OVA peptide was first optimised for this reaction. Past literature recommends concentrations ranging from 10µg/ml (Li et al., 2009, Rafiq et al., 2002) to 50µg/ml (Huang et al., 2010, Bedoret et al., 2009). Both doses of OVA peptide were incubated with OT-‐1 splenocytes for 72 hours and the percentage of proliferated CD8 T cells was quantified using flow cytometry. Unstimulated cultures failed to proliferate (Figure 3.12A), with 0.32±0.1% of CD8 cells losing CTV fluorescence (Figure 3.12D). Cultures stimulated with 10µg/ml and 50µg/ml had significantly more CD8 T cell proliferation compared to controls, but there was no significant difference between the doses (32.3±2.1% vs. 32.0±3%; Figure 3.12B, C, D). Therefore, 10µg/ml OVA peptide dose was chosen for future experiments.
Figure 3.12 | Optimisation of OVA peptide dose with OT-‐1 splenocytes. CTV fluorescence histograms of CD8 T cells in OT-‐1 splenocyte cultures supplemented with (A) no OVA peptide (n=4), (B) 10µg/ml OVA peptide (n=2), and (C) 50µg/ml OVA peptide (n=4). (D) Quantification of CD8 T cell proliferation. Data shown as mean±SD. Statistical analysis performed using One-‐way ANOVA with Bonferroni’s Multiple Comparison Test. In all experiments, n is indicative of technical repeats using the same culture of PαS MSCs.
I then optimised the culture time to achieve maximal CD8 T cell proliferation. As the OVA peptide needs to be processed and presented, there will be a lag phase before T cell proliferation. For example, co-‐culture with Dynabeads® for 72 hours caused a proliferative response in >90% of CD8 T cells (Figure 3.11D), while <35% divided when co-‐cultured with OVA peptide for the same time period (Figure 3.12D). The effect of 10µg/ml OVA peptide stimulation for 3, 4 and 6 days culture was examined. Significant increases in CD8 T cell proliferation were observed at days 4 and 6 compared to day 3 cultures (Figure 3.13).
However, there was no significant difference between the CD8 T cell proliferation seen at day 4 (93.6±0.7%) compared to day 6 (94.4±1.1%). Flow cytometry histograms for all time-‐ points alongside their matched controls are displayed in Figure 3.14. As such, 4-‐day cultures supplemented with 10µg/ml OVA peptide was used to stimulate OT-‐1 CD8 T cell proliferation and to examine the effect of C57BL/6-‐derived PαS MSC co-‐culture.
Figure 3.13 | Optimisation of overall culture period with OT-‐1 splenocytes. OT-‐1 cells were cultured unstimulated or stimulated with 10µg/ml OVA peptide for three (n=5), four (n=5), or six (n=6) days. The percentage of proliferated CD8 T cells was then quantified using flow cytometry based on CTV fluorescence. Data shown as mean±SD. Statistical analysis performed using One-‐way ANOVA with Bonferroni’s Multiple Comparison Test. In all experiments, n is indicative of technical repeats using the same culture of PαS MSCs.
Figure 3.14 | CTV fluorescence histograms of OT-‐1 CD8 T cells cultured for different periods of time. No proliferation was observed in unstimulated control cultures at days three (A; n=4), four (B; n=6), and six (C; n=3). Limited cell division was seen in stimulated cultures at day three (D; n=5), but over 90% CD8 T cell proliferation was seen at day 4 (E; n=5) and 6 (F; n=6). In all experiments, n is indicative of technical repeats using the same culture of PαS MSCs.