La economía circular

T cell proliferation is typically used as a model of immune cell activation. Upon presentation with an antigen or a stimulatory signal, T cells undergo clonal expansion and cell differentiation48_{. Apoptosis of these cells is a crucial process to maintain homeostasis following}

immune stimulation49_{. In this study, the effect of red blood cells on the proliferation and}

survival of T cells was evaluated. Most literature in this area has been focused on T cells, although a handful of papers have also looked at B cells and dendritic cells. The experiments in this chapter were performed on a leukemic T cell line (Jurkat cells) and on a mixed cell population of freshly isolated PBMCs. Analysis of this complex cell population was done to more closely model what may be occurring in vivo. However, in such models, it is difficult to elucidate the role and effect of the different cell types engaged in crosstalk. As such, where cytokines are the primary analytes, there would be value in the analysis of isolated lymphocyte populations in contact with red blood cells. Transcriptomics would be an alternative method, which may prove useful in the analysis of mixed populations such as PBMCs after stimulation

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with red blood cells. Prior to RNA extraction the mixed population could be rapidly and gently separated using magnetic-activated cell sorting, which is available from a number of suppliers. 5.4.2.1 Jurkat cell proliferation: treatment with intact red blood cells

Jurkat cells are a leukemic T cell line that are frequently used to model T cell activity. Increased proliferation of these cells is used as an indication of stimulation with an immunogenic agent. This study demonstrated that upRBCs stimulated the proliferation of Jurkat cells by approximately 2-fold (Figure 5.4). Of the few publications investigating the effect of healthy red blood cells on T cell proliferation, one looked at the effect on Jurkat cells9_{. This study}

observed an approximately 5-fold increase in the proliferation of Jurkat cells in the presence of naïve red blood cells. The differences between the two studies here may be an artefact of red blood cell storage prior to analysis. In the study described in this chapter, the red blood cells were stored at 37 °C for three days before analysis, whereas in the published study, the red blood cells were stored at 4 °C for a non-specified period of time. Long term storage at 4 °C has been demonstrated to affect the capacity of red blood cells to stimulate proliferation of T cells19,50_{, as such, short term storage at 4 °C or otherwise may have also had an effect on the}

level of T cell proliferation following treatment with those red blood cells.

Jurkat cells treated with red blood cells primed with cancer cells (pRBC) proliferated significantly more than the upRBC group (Figure 5.4). A549 cells were incubated with two different concentrations of red blood cells (low and high) to produce two groups of primed cells (pRBC-L, or pRBC-H respectively). Both groups of primed cells stimulated increased proliferation of the Jurkat cells, however, this effect was most pronounced in the pRBC-L group. This group stimulated 8.3-fold more proliferation than the untreated controls, whilst the pRBC-H groups stimulated only 5.3-fold more proliferation than the untreated controls (Figure 5.4). It is unclear as to why the pRBC-L group was more immunogenic than the pRBC-H group, but it may be a result of the concentration of available soluble factors secreted from the A549 cells being diluted across more red blood cells or simply an artefact of the static culture conditions. Fonseca et al. demonstrated that the activity of red blood cells was reliant upon them being intact and in direct contact with the immune cells14_{. Whilst the red blood cells were}

being primed by the cancer cells, they were left to settle to the bottom of the flask, and thus were in constant contact with the A549 cells. In the pRBC-H group, the layer of red blood cells was much more substantial than in the pRBC-L group and so the amount of direct contact between the cells may be have reduced. In turn, this may have reduced the immunogenic potential of these red blood cells. Following these data, the pRBC-L group was chosen as the

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optimal group for stimulating T cell proliferation through cancer cell priming. All of the following T cell experiments were performed on the pRBC-L primed group only.

5.4.2.2 Jurkat cell proliferation: treatment with red blood cell lysate, cytosols, or membranes

Jurkat cells treated with red blood cell components including lysates, cytosols, or membranes did stimulate increased proliferation (Figure 5.4), with no significant difference observed between the upRBC and pRBC groups for the lysate and cytosol samples. However, this was not the case for the red blood cell membranes. The membranes isolated from pRBCs stimulated the Jurkat cells to proliferate significantly more than the membranes of the upRBCs with a mean fold change of 1.9 and 1.4 respectively (Figure 5.4). These results demonstrate that priming red blood cells with cancer cells resulted in a modification of the red blood cell membrane that made the membranes more immunogenic. This result is not surprising, as some inflammatory cytokines are known to bind to specific receptors on the red blood cell membrane51,52 _{and upon lysis both sides of the membrane would be available for binding,}

which may have resulted in an increase in non-specific binding.

5.4.2.3 Jurkat cell proliferation: treatment with red blood cell conditioned media The conditioned media from A549 cells alone stimulated the Jurkat cells to proliferate significantly more than the media blank control with a fold change of 3.5 compared to 1.2-fold for the media control (Figure 5.4). Of note, the conditioned media from the red blood cells and the A549 cells incubated together stimulated the Jurkat cells to proliferate significantly more (3.5-fold) than the conditioned media of either component alone (2.9-fold for A549 cells, 1.5- fold for red blood cells) (Figure 5.4). It has been previously demonstrated that the immunogenic activity of naïve red blood cells was mediated by soluble protein factors and that these factors were present in the non-vesicle fraction of the red blood cell conditioned media9_{. The increase}

in proliferation observed with the conditioned media in this study was a result of a change in one or more soluble factors and this change was dependent upon the inclusion of red blood cells in the culture. Together, the results of these experiments support the observation that a soluble factor may be mediating the stimulatory activity of red blood cells.

Antunes et al. reported that conditioned media from red blood cells alone stimulated the Jurkat cells to proliferate as much as the intact red blood cells9_{, however, this result was not replicated}

in the study outlined in this chapter. No significant difference was observed between treatment with red blood cell conditioned media and the media control (Figure 5.4). There are a few

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differences between the studies that may explain why no further proliferation was observed. In the study outlined by Antunes et al., they used 6x more red blood cells to produce the conditioned media and the red blood cells were incubated in a serum-free media. In contrast, in this study the red blood cell conditioned media was produced in a culture media that contained 10 % FBS. This high level of FBS and the low level of red blood cells is likely that reason that no difference was observed between the red blood cell conditioned media and the corresponding media blank.

5.4.2.4 Jurkat cell cytokine release: treatment with intact red blood cells

The level of cytokines in Jurkat conditioned media was increased following treatment with pRBCs compared to upRBCs, with 14 cytokines present at significantly higher levels in the pRBC group (Section 5.3.3.2). Unstimulated Jurkat cells secrete minimal inflammatory cytokines (Figure 5.5). Treatment with red blood cells was sufficient to stimulate the cells to secrete a number of cytokines. As observed with Jurkat cell proliferation, treatment with pRBCs produced a different profile than upRBCs. This demonstrates that the red blood cells were functionally altered as a result of priming. Whilst the results of the Jurkat cells are interesting, they are limited in their ability to model normal immune activity. Inactivated, these cells typically do not secrete many cytokines, and when they are activated, the signalling pathways become dysregulated53_{. For this reason, more biologically relevant analyses were}

performed using fresh peripheral blood mononuclear cells.

5.4.2.5 PBMC proliferation: treatment with intact red blood cells

In a mixed cell model, proliferation of T cells (CD3+) and subsets of T cells (CD4+ and CD8+ cells) were evaluated using CFSE staining. A small number of papers have investigated the effect of red blood cell treatment on the proliferation of cells in a PBMC population. In these papers, a consistent increase in proliferation of PBMCs following treatment with naïve red blood cells has been reported9,10,12–15_{. However, as there are so few papers reporting the}

proliferation of fresh T cells treated with red blood cells, there is no uniform set of methods that are used. As such, comparison of results between the papers was challenging.

In the study outlined in this chapter, cell proliferation was significantly increased with pRBC treatment for CD3+, CD4+, and CD8+ cells (Figure 5.8). Treatment with pRBCs was demonstrated to stimulate an increase in the proliferation of CD8+ cells but not CD4+ cells (Figure 5.8). In a CD3+ population, the number of CD4+ cells, unlike CD8+ cells, did not change with phytohemagglutinin (PHA-P) stimulation or with PHA-P stimulation and red

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blood cell treatment (Figure 5.9). The number of CD8+ cells increased significantly with red blood cell treatment (Figure 5.9). This increase in the number of cytotoxic T cells (CD8+ cells) following red blood cell treatment has been reported previously in both PBMC cultures and purified CD3+ cultures11,12_{. It is not clear if the reason for this selective expansion of CD8+}

cells in vitro is simply a result of a faster doubling time than CD4+ cells or if it was true preferential expansion. These results were obtained in an in vitro model, which comes with its own limitations. In this study, the proportion of red blood cells to lymphocytes was 1:10, which is well out of the range of the normal ratio (1:3000). However, this was not viable for an in vitro study. In addition, the experiments were performed over a restricted time frame (5 days), which may have limited the overall CD4+ cell proliferation. Patients receiving multiple red blood cell transfusions, a similar preferential expansion of CD8+ cells has been observed54,55_.

In one study, participants receiving multiple transfusions for sickle cell anaemia or haemophilia had significantly higher levels of CD8+ T cells than the non-transfused controls54_{. It has been}

suggested that this preferential CD8+ cell proliferation may be one of the reasons for the immunosuppression that is observed following red blood cell transfusions8_{, however more}

work is required to investigate this hypothesis. In addition, the cause and effect of the increased proliferation of the CD8+ cells with primed red blood cell treatment of PBMCs observed in this study warrants further investigation.

5.4.2.6 PBMC survival and apoptosis: treatment with intact red blood cells

PHA-P is a stimulant for PBMC proliferation, however treatment with PHA-P also stimulates apoptosis in those same cells. In this study, the number of apoptotic and live cells was determined by assessing Annexin V staining of T cell subsets. The percentage of live cells dropped significantly from a mean of 73 % or 81 % for CD4+ and CD8+ cells respectively in the untreated group, to 46 % in the PHA-P stimulated group for both CD4+ and CD8+ cells (Figure 5.10). Treatment with red blood cells (either upRBCs or pRBCs) protected the CD4+ and CD8+ cells from PHA-P induced apoptosis (Figure 5.10). This protective activity of red blood cells on PBMCs has been well documented in the literature9,12,15_{. It has been shown that}

this promotion of cell survival in response to red blood cells is reliant on direct cell to cell contact and is specific to T cells and is independent of monocyte activity14_.

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