miRNA-based secretome therapeutics (SYN, TA1, IA1 and IA2) have been successfully produced in our laboratory based on allorecognition (± polymer-mediated immunocamouflage) based MLR. (3, 4, 25) In order to compare the effects of our acellular secretome products to mitogen or monoclonal antibody mediated pan T cell activation and alloactivation, cell proliferation and CD4+/CD8+ subset composition among the CD3+ population were measured (Figure 2). As shown, resting PBMC demonstrated minimal proliferation (1.5 ± 0.4%) and a CD4:CD8 ratio of 1.7 ± 0.1. In contrast, the pan T cell activators anti-CD3/CD28 and PHA induced massive CD3+ cell proliferation (78.1 ± 1.8% and 94.4 ± 0.3%, at days 3 and 4, respectively, p < 0.0001) and altered the CD4:CD8 ratio (Figure 2A). Within the proliferating CD3+ T cells, a significant increase (1.8% to 6-7%) in CD4+CD8+cells were noted. These poorly studied cells have been speculated to play a role in autoimmune and chronic inflammatory disorders.(38) Despite both PHA and anti-CD3/CD28 being Pan T cell activators, there were differences in how these agents modulated the CD4/CD8 differentiation. PHA, but not anti-CD3/CD28, significantly increased the CD8+ population while simultaneously decreasing the CD4+ population resulting in a significant (p < 0.0001) decrease of CD4:CD8 ratio relative to resting PBMC (1.7 to 0.9). mAb activation also decreased the ratio but not as dramatically as PHA. Alloactivation, in comparison to the highly potent pan T cell activation, induced a more moderate proliferation of CD3+ cells (30.9 ± 3.4% at Day 10; p < 0.0001) relative to resting PBMC. The reduction in proliferation arose consequent to <10% of T cells within a population typically being capable of allorecognition (Figure 2B). (39, 40) Nonetheless, alloactivation similarly increased the CD4+CD8+ population and decreased CD4+ cells leading to a significantly (p < 0.01) decreased CD4:CD8 ratio.
The secretome products showed significant variability (at Day 10) in their effects when used to activate resting PBMC. As expected, resting PBMC treated with the SYN secretome were virtually identical to the resting PBMC with regards to both proliferation, subset analysis and CD4:CD8 ratio. Similarly, the tolerogenic TA1 preparation showed minimal proliferation and no substantive changes in the subset differentiation or the CD4:CD8 ratio. Of note, the small increase in CD3+ proliferation (2.3 ± 0.1%) supports earlier observations showing increased proliferation/differentiation of Treg (CD4+Foxp3+) cells in TA1-treated PBMC.(21) However, in contrast to the SYN and TA1 products, IA1 and IA2 showed significant variation from both the resting PBMC and from each other (Figure 2C). IA1, derived from a control MLR, significantly increased the total CD3+ cell proliferation (12.2 ± 1.2%, p < 0.001) and decreased the relative abundance of CD4+ cells (38.8 ± 3.4% versus 56.0 ± 1.5% for resting PBMC). Consequent to this change, the IA1 CD4:CD8 ratio was significantly reduced (1.0 ± 0.1; p < 0.001) suggestive of a pro-inflammatory state and similar to that noted with the pan T cell activators. Interestingly, the cancer cell (HeLa) stimulated biologic IA2 while inducing a similar level of CD3+ cell proliferation (10.7 ± 0.5%, p < 0.01), showed dramatically different phenotype distribution (Figure 2C). In contrast to IA1 which reduced the CD4:CD8 ratio, IA2 significantly increased the ratio relative to both IA1 and the resting PBMC (2.1 ± 0.4, 1.0 ±0.1, and 1.7 ± 0.1; respectively) consequent to a decrease in CD8+ cells and a dramatic increase (p<0.05) in CD4-CD8- cells. These double negative cells, while poorly studied, have been implicated in both inflammation and as regulatory cells.(41) Interestingly, our previous study suggested that the anti-HeLa effects of IA2 treated PBMC were different from that of IA1 treated PBMC. (4, 25)
To further investigate the effects of the secretome products, CD4+ Th1, Th17, Treg and CD3+6B11+ iNKT cell were examined 10 days post activation and compared to a human MLR (Figure 3A). As expected, the SYN product had minimal effect on the differentiation of any subset relative to the resting PBMC (grey bar; a) while the tolerogenic TA1 product increased Treg cells (Figure 3A).(21) In contrast, the TA1, IA1 and IA2 secretome products variably affected the immune cell subsets. Most notably, IA1 very significantly increased Th17 cells and, to a lesser extent, Th1 and iNKT cells. The IA1-mediated increase in Th17 cells was similar to that observed within an MLR. Further showing the biological disparity between the IA1 and IA2 products, IA2 exhibited less effect on Th17 and Th1 cells but a greater effect on iNKT cells. Consequent to the differential changes within the T cells, the Th17:Treg ratio was affected (Figure 3B). As shown the SYN product had no effect while TA1 slightly increased the ratio while IA1 significantly increased the ratio similar to that seen in an MLR; perhaps indicative of the preparation of IA1 from the MLR secretome. IA2 slightly, but not significantly, increase the ratio – perhaps due to the expansion of the CD4-CD8- population (Figure 2C). Due to the evolutionary conservation of miRNA, the cross-species efficacies of the human and murine sourced secretomes on murine splenocytes was examined relative to control splenocytes (grey bar; a) and a murine MLR (Figure 3C-D). As shown, human sourced SYN exerted no effect on the levels of Th17 and Treg cells relative to resting splenocytes. In contrast, both human and murine IA1 preferentially depressed Treg cells relative to Th17 cells (Figure 3C). This resulted in a significant increase in the Th17:Treg ratio for both human (p< 0.05) and murine (p<0.0001) IA1 and similar to that seen in a murine MLR (Figure 3D). Hence, these data demonstrated that the different secretomes (SYN, TA1, IA1 and IA2) exerted differential effects on T cell subsets but that the evolutionarily conserved miRNA composition of the human and murine IA1 exhibited cross species efficacy.
To determine how these differential T cell activation strategies (i.e., Pan T cell, Allo and Secretome) affected resting human PBMC, intracellular miRNA expression was measured 72 hours post activation and the expression of 84 miRNA involved in immunopathology pathways were assess profiled via clustergram, Log2 fold change and volcano plot analyses. As shown in Figure 4A, clustergram analysis demonstrated that PBMC activation by pan T cell activators (PHA and anti-CD3/CD28; P) resulted in significantly different miRNA expression profiles relative to resting (i.e., unactivated) PBMC with a general trend towards a complete inversion (i.e., green to red and red to green) of the miRNA signals. Alloactivation (Allo) also induced distinct miRNA expression profiles relative to resting PBMC, but to a lesser extent than pan T cell activators. Moreover, the Control MLR and mPEG-MLR, from which IA1 and TA1 are derived, showed similarities in the overall pattern of miRNA expression but some distinct differences in specific miRNA that results in dramatically different biological responses. (3, 4, 25) In comparison to pan T cell and allo activations, the secretome products (S) induced miRNA expression patterns that were very distinct from that of the Pan T cell activators.(4, 25) To further illustrate differences between pan T cell (P) and secretome (S) activation, the log2 fold change of thirteen differentially expressed miRNA, as well as the relative patterns of expression, are shown (Figure 4B). These miRNA were selected from the clustergram analysis as well as previous studies and their putative/described functions are summarized in Table 1. (3, 4, 25) It is important to note however, that the ‘putative’ functions of the distinct miRNA can vary significantly depending on the model used and are, typically, best estimates of their function in the absences of examining the miRNA expression pattern as a whole. Based on the low fidelity of miRNA, perhaps the most informative approach, is the analysis of the differential effects that these agents had on the relative pattern of miRNA expression (Figure 4C). As noted, both PHA and anti-CD3/CD28 showed similarities in their patterns of expression and in the extent of proliferation induced (Figure 2; 94.4 ± 0.3 and 78.1 ± 1.8%, respectively). In contrast, the secretomes induced a more subtle change in miRNA expression. The IA1 secretome pattern of expression was the most similar to the pan-T cell activators – though it differed significantly in the magnitude of changes in miRNA expression and T cell proliferation (12.2 ± 1.2%). In contrast, the tolerogenic TA1 secretome pattern varied significantly from IA1 and most closely resembled the miRNA expression of unactivated PBMC. IA2 similarly varied significantly from IA1 despite inducing a similar level of T cell proliferation (10.7 ± 0.5%). Importantly, the differential patterns of expression of these miRNA was also associated with differential biological effects with TA1 inducing systemic tolerance in a murine model and IA1 enhancing PBMC-mediated inhibition of cancer cell growth and IA2 exhibiting direct toxicity (apoptosis) of cancer cells.(3, 4)
To further compare the miRNA expression profile of IA1 relative to the pan-T cell (anti-CD3/CD28) activation and the TA1 and IA2 secretomes, volcano plot analyses were conducted (Figure 5). Volcano plot analyses visualizes the miRNA data based on log scale changes and allows for statistical comparison of the expression of discreet miRNA between samples – but largely misses out on the overall PATTERN of changes. As noted in Figure 5A, distinct differences are noted between IA1 and anti-CD3/CD28. IA1 significantly (p < 0.05) upregulated the expression of miR-125b-5p and miR-451a relative to anti-CD3/CD28, while miR-18a-5p, miR-17-5p, miR-20a-5p and miR-135b-5p were downregulated. Similar to Figure 4 multiple other miRNA were also differentially expressed between IA1 and anti-CD3/CD28 activation though they did not reach significance in the volcano plot analyses (though if compared to resting PBMC they are different). Interestingly, the miRNA expression profiles between IA1 and TA1 were not statistically significantly different (Figure 5B), though, as also seen in Figure 4, miR-298, miR-214-3p, miR-302a-3p and miR-206 were over-expressed in IA1 relative to TA1. Finally, the expression of miR-149-5p and miR-18b-5p were significantly (p < 0.05) upregulated in PBMC treated with IA1 when compared to the same donor PBMC treated with IA2 (Figure 5C).
In sum, clustergram, log2 fold change and volcano plot analyses demonstrate the differential activation strategies yielded dramatically different miRNA expression profiles that in turn resulted in significant differences in T cell activation and subset differentiation. In order to better understand these differences, an integrative Venn diagram analysis was done using all three sets of data (Figure 6) in order to differentially compare the pan T cell, allorecognition and secretome activation. As demonstrated, Pan T cell activation using PHA and anti-CD3/CD28 yielded similar, though not identical, changes in miRNA expression (solid circles = over expression; dashed circles =reduced expression; overlap are miRNA in common). For further comparison purposes, we averaged the miRNA expression profile and proliferation rates of PHA and anti-CD3/CD28 to represent the efficacy of pan T cell activation strategy. In contrast to Pan T cell activation, the miRNA changes induced by allorecognition were much more discreet (relative to resting PBMC) and highly limited when compared to the Pan T cell activators. Moreover, allorecognition resulted in a significant reduction in cell proliferation (Pan T: 86.3% versus 30.9% for Allorecognition). Similar to the allorecognition response, the allo-derived IA1 secretome also reduced the miRNA response pattern relative to Pan T cell activation and, not surprisingly, was similar to the pattern of expression observed in the alloresponse but with the increased expression of miR-298 and decreased expression of miR-206 and miR-214-3p. While some miRNA are in common to the Pan T cell, Allo, IA1 and IA2 pro-inflammatory responses (overlaps in Venn diagrams), some of these (e.g., miR-155-5p) are also implicated in the tolerogenic TA1 and mPEG-Allo responses. This again argues that the ‘pattern of miRNA expression’ (Figure 4C) encompassing increases, decreases and static levels of multiple, rather than a specific (single or small number), of miRNA is crucial.
Importantly, the biomanufaturing process is of importance. This is most obvious in comparing the MLR vs mPEG-MLR miRNA patterns, but it is also observed within the pro-inflammatory IA1 and IA2 secretome products (Figure 7). While IA1 and IA2 stimulate similar proliferative effects (12.2 ± 1.2 and 10.7 ± 0.5%; Figure 2), their biological activity was dramatically different. As shown in Figure 7A, control HeLa cells treated with either IA1 or IA2 secretome-activated PBMC showed similar inhibitory effects on HeLa cell proliferation over 7 days as evidenced by quantification of Area Under Curve (AUC). However, the effect of IA2 was not predicated on the presence of PBMC. When the seeded HeLa cells were overlaid with the secretomes themselves (absent PBMC), IA2 exerted direct inhibitory effects on HeLa cell proliferation while SYN, TA1 and IA1 had no inhibitory effects (Figure 7A). Indeed, PBMC pre-treated with IA1 and IA2 induced entirely different miRNA expression (Figure 7B) that resulted in vastly different responses to the HeLa cells. As shown in Figure 7C, the IA1 treated PBMC resulted in a significantly enhanced cell:cell interaction between the PBMC and HeLa cells following 72 hours co-culture. In contrast, minimal cell:cell interaction of IA2-treated PBMC with the HeLa cells was noted at 72 hours, though the HeLa cells themselves showed evidence of apoptotic/necrotic blebbing suggestive of non-contact dependent killing. The same morphological picture was observed with the direct overlay of IA2 in the absence of PBMC. These findings agreed with our previous hypothesis that IA2 inhibited cancer cells proliferation via non-PBMC-mediated pathways and/or apoptotic mechanisms. (4)
In sum, these studies demonstrate that pan T cell activators, alloactivation and secretome therapeutics exerted differential efficacies on leukocyte proliferation/differentiation and miRNA expression. Pan T cell activators induced massive T cell proliferation and miRNA alteration profiles relative to resting cells. Allo-MLR demonstrated a more discriminatory activation of resting PBMC relative to pan T cell activators, while mPEG-MLR diminished allorecognition. Importantly, IA1 and TA1 secretome derived from allo- and mPEG-MLR respectively, exerted similar immunomodulatory efficacies to its origin MLR response. In contrast, resting cell generated secretome SYN had minimal effects on recipient resting PBMC. Of interest, The HeLa-MLR derived IA2 therapeutic exhibited distinct alterations to the leukocyte miRNA expression profile, suggesting an apoptosis-mediated immunomodulatory and anti-cancer pathway.