**3. Immunomodulation via immunocamouflage and differential miRNA production**

Previous studies from our laboratory demonstrated that a persistent and systemic reorientation of the animal (murine; or *in vitro* human) immune response towards a tolerogenic response could be induced via the adoptive transfer of immunocamouflaged allogeneic leukocytes to a recipient animal [32–43]. Immunocamouflage of cells is mediated by the covalent grafting of methoxypoly(ethylene glycol) (mPEG) to the leukocyte membrane surface. Consequent to mPEG-grafting (PEGylation), MHC-mediated T cell alloproliferation is dramatically inhibited due to consequent to impaired cell:cell interaction and weak allostimulation (**Figure 1A** and **B**). These studies demonstrated that the PEGylated allogeneic leukocytes diminished intracellular communication preventing a Teff response while simultaneously inducing the generation of Treg cells skewing the Treg:Teff ratio towards a tolerogenic state (**Figure 1B** and **C**) [36, 38–43]. Further *in vitro* and *in vivo* studies demonstrated that, using MLR-based secretome biomanufacturing systems, distinct acellular microRNA (miRNA) based therapeutics could be manufactured from control and PEGylated allorecognition reactions that systemically and persistently reorient the immune response to either a proinflammatory (IA1) or tolerogenic (TA1) state (**Figure 1**) [40, 43]. In this chapter, we will demonstrate how these miRNA-based therapeutics can inhibit the progression of T cell mediated autoimmune diseases (TA1) or conversely enhance the proinflammatory anticancer T cell response (IA1).

### **4. Production of miRNA therapeutics via the alloresponse pathway**

Since their discovery in 1996, the role of circulating (cell-free) miRNA in disease processes has become an active research area and recent findings suggest that they may be biomarkers, or possibly mediators, of cancers as well as autoimmune diseases such as T1D [44–46]. To understand mechanistically how the TA1 and IA1 miRNA biologics function, an appreciation of the biological role and regulatory complexity of miRNA is needed. Recent studies have demonstrated that miRNA are key epigenetic regulators of cellular processes including immune responses, inflammation, proliferation, survival, and cellular differentiation [47, 48]. miRNA are short (~22 nucleotides) single-stranded RNA molecules found in all eukaryotes and it is estimated that ~60% of mammalian genes are targeted by one or more miRNA [49, 50]. Moreover, because of their evolutionary importance in gene regulation, miRNA and their

**77**

**Figure 2.**

*Modulating the T Lymphocyte Immune Response via Secretome Produced…*

sequence and processing are highly conserved between mammalian species

(e.g., mouse and human) [49]. While miRNA are most commonly found intracellularly, significant amounts of stable miRNA are also found in the serum of mammals suggesting an important messenger/regulatory role. While the nomenclature of miRNAs is relatively straightforward it is important to note that similarities in miRNA number designation is not indicative of similarity in functionality (**Figure 2**). Moreover, the literature is replete with conflicting claims for the specific actions of a single miRNA. Indeed, there is a significant lack of clarity regarding the function of a single miRNA. This lack of functional clarity likely arises consequent to the complexity and low fidelity of the miRNA bioregulatory process. Of note, a single miRNA can potentially affect tens to hundreds of genes and individual genes can be regulated by multiple miRNA [50]. Hence, the effect of modifying the expression of a single miRNA on protein regulation and bioregulatory networks is unpredictable. Because of this regulatory complexity, most studies have focused on miRNA as disease biomarkers, not as therapeutic agents as there is a low probability that altered expression of a single, or even a few, miRNA would exert a potent and definitive biological response [51–54]. From a bioregulatory approach, it is more probable that multiple miRNA control protein expression, proliferation and differentiation and it is this "pattern of miRNA expression" (encompassing increased, decreased and static levels) that must be mimicked to achieve pharmacologically effective miRNAbased therapeutics. To achieve this goal our laboratory approach has been purposefully chosen to biologically manufacture relatively complex miRNA preparations mimicking normal biology in order to achieve maximal biological functionality. Using a Mixed Lymphocyte Reaction (MLR) production model the T cell centric proinflammatory IA1 and tolerogenic TA1 therapeutics can be reproducibly manufactured using the control-MLR and mPEG-MLR (respectively; **Figure 1**). As demonstrated, the allogeneic PBMC populations within the control- and mPEG-MLR express significantly different patterns of miRNA expression relative to resting

*miRNA nomenclature explained. An important concept to understand is that the miRNA number (e.g., 123 as shown) has no relationship to function or structure. For example, "hsa-miR-123" has no implied structural or functional similarity to "hsa-miR-128." However, because of the highly conserved nature of miRNA, human* 

*"hsa-miR-123" has very similar structure and function to murine "mmu-miR-123."*

*DOI: http://dx.doi.org/10.5772/intechopen.86598*

#### *Modulating the T Lymphocyte Immune Response via Secretome Produced… DOI: http://dx.doi.org/10.5772/intechopen.86598*

*Cells of the Immune System*

**production**

therapies are currently focused on the *ex vivo* expansion and subsequent transfusion of autologous Teff capable of killing the cancer cells [23–31]. However, these current immune enhancing methods, while promising, are expensive, complicated to accomplish (e.g., insertion of specific target cancer genes in APC) and requires

Perhaps most importantly, current tolerogenic or proinflammatory therapeutic approaches fail to persistently reorient the systemic T cell immune response thus necessitating continual therapy. Moreover, despite the importance of the Treg:Teff ratio, in both autoimmune diseases and cancer, there are a paucity of pharmacologic tools that can directly, and in tandem, target the regulation of both the Treg and Teff subsets. Hence, to diminish or overcome the need for chronic administration of immunotherapeutic agents, new approaches capable of persistently reorienting the

**3. Immunomodulation via immunocamouflage and differential miRNA** 

Previous studies from our laboratory demonstrated that a persistent and systemic reorientation of the animal (murine; or *in vitro* human) immune response towards a tolerogenic response could be induced via the adoptive transfer of immunocamouflaged allogeneic leukocytes to a recipient animal [32–43]. Immunocamouflage of cells is mediated by the covalent grafting of methoxypoly(ethylene glycol) (mPEG) to the leukocyte membrane surface. Consequent to mPEG-grafting (PEGylation), MHC-mediated T cell alloproliferation is dramatically inhibited due to consequent to impaired cell:cell interaction and weak allostimulation (**Figure 1A** and **B**). These studies demonstrated that the PEGylated allogeneic leukocytes diminished intracellular communication preventing a Teff response while simultaneously inducing the generation of Treg cells

skewing the Treg:Teff ratio towards a tolerogenic state (**Figure 1B** and **C**) [36, 38–43]. Further *in vitro* and *in vivo* studies demonstrated that, using MLR-based secretome biomanufacturing systems, distinct acellular microRNA (miRNA) based therapeutics could be manufactured from control and PEGylated allorecognition reactions that systemically and persistently reorient the immune response to either a proinflammatory (IA1) or tolerogenic (TA1) state (**Figure 1**) [40, 43]. In this chapter, we will demonstrate how these miRNA-based therapeutics can inhibit the progression of T cell mediated autoimmune diseases (TA1) or conversely enhance the proinflammatory anticancer T cell response (IA1).

**4. Production of miRNA therapeutics via the alloresponse pathway**

Since their discovery in 1996, the role of circulating (cell-free) miRNA in disease processes has become an active research area and recent findings suggest that they may be biomarkers, or possibly mediators, of cancers as well as autoimmune diseases such as T1D [44–46]. To understand mechanistically how the TA1 and IA1 miRNA biologics function, an appreciation of the biological role and regulatory complexity of miRNA is needed. Recent studies have demonstrated that miRNA are key epigenetic regulators of cellular processes including immune responses, inflammation, proliferation, survival, and cellular differentiation [47, 48]. miRNA are short (~22 nucleotides) single-stranded RNA molecules found in all eukaryotes and it is estimated that ~60% of mammalian genes are targeted by one or more miRNA [49, 50]. Moreover, because of their evolutionary importance in gene regulation, miRNA and their

weeks of tissue culture expansion to meet the threshold for cell infusion.

endogenous immune (Treg:Teff) response would be of value.

**76**

sequence and processing are highly conserved between mammalian species (e.g., mouse and human) [49]. While miRNA are most commonly found intracellularly, significant amounts of stable miRNA are also found in the serum of mammals suggesting an important messenger/regulatory role. While the nomenclature of miRNAs is relatively straightforward it is important to note that similarities in miRNA number designation is not indicative of similarity in functionality (**Figure 2**). Moreover, the literature is replete with conflicting claims for the specific actions of a single miRNA.

Indeed, there is a significant lack of clarity regarding the function of a single miRNA. This lack of functional clarity likely arises consequent to the complexity and low fidelity of the miRNA bioregulatory process. Of note, a single miRNA can potentially affect tens to hundreds of genes and individual genes can be regulated by multiple miRNA [50]. Hence, the effect of modifying the expression of a single miRNA on protein regulation and bioregulatory networks is unpredictable. Because of this regulatory complexity, most studies have focused on miRNA as disease biomarkers, not as therapeutic agents as there is a low probability that altered expression of a single, or even a few, miRNA would exert a potent and definitive biological response [51–54]. From a bioregulatory approach, it is more probable that multiple miRNA control protein expression, proliferation and differentiation and it is this "pattern of miRNA expression" (encompassing increased, decreased and static levels) that must be mimicked to achieve pharmacologically effective miRNAbased therapeutics. To achieve this goal our laboratory approach has been purposefully chosen to biologically manufacture relatively complex miRNA preparations mimicking normal biology in order to achieve maximal biological functionality.

Using a Mixed Lymphocyte Reaction (MLR) production model the T cell centric proinflammatory IA1 and tolerogenic TA1 therapeutics can be reproducibly manufactured using the control-MLR and mPEG-MLR (respectively; **Figure 1**). As demonstrated, the allogeneic PBMC populations within the control- and mPEG-MLR express significantly different patterns of miRNA expression relative to resting

#### **Figure 2.**

*miRNA nomenclature explained. An important concept to understand is that the miRNA number (e.g., 123 as shown) has no relationship to function or structure. For example, "hsa-miR-123" has no implied structural or functional similarity to "hsa-miR-128." However, because of the highly conserved nature of miRNA, human "hsa-miR-123" has very similar structure and function to murine "mmu-miR-123."*

PBMC as evidenced via clustergram (**Figure 3A**), volcano plot (**Figure 3B**) and Log2 Fold (**Figure 3C**) miRNA expression analyses. Importantly, as shown in **Figure 3C**, the control- and mPEG-MLRs show unique patterns of expression. While there are some similarities in the pattern of expression there are significant disparity in miRNAs expressed as well (not shown are the miRNA unchanged from resting cells).

Importantly, the differences in miRNA expression between the Control- and mPEG-MLR leukocyte yield secretomes that exert dramatically different effects when used to treat resting human PBMC or murine splenocytes. Collection of the secretome produced (**Figure 4A**) during the control and polymer modified allorecognition-based MLR yields a reproducible, acellular, miRNA-rich, material that is stable and can be frozen and thawed with minimal decrement to its activity. As schematically presented (**Figure 4B**), TA1 upregulates regulatory T cell populations (e.g., Treg) while simultaneously downregulating Teff (e.g., Th17 and Th1) cells. In contrast, the proinflammatory IA1 increases Teff while decreasing Treg cells. Of note, the secretome from resting cells (SYN) has minimal to no effect on human or mouse immune cells. Moreover, due to the conserved nature of mammalian miRNA, cross species efficacy is observed with both TA1 and IA1. As shown in **Figure 4C**, murine splenocyte produced TA1 and IA1 exerted dose-dependent effects on a human MLR with murine-sourced TA1 reducing CD3+ CD4+ T cell proliferation and the murine IA1 enhancing CD3+ CD4+ T cell proliferation. Hence, a polymer-based, alloresponse manufacturing system may provide a unique avenue for more effectively, and safely, modulating the Treg:Teff cell ratio via the production of therapeutically effective TA1 and IA1 miRNA-based therapeutics [32–43]. Importantly, the effects of TA1 and IA1 immunotherapy was persistent. In murine studies, a single dosing of TA1 to mice resulted in significant increase in Treg cells within the spleen of normal mice that persisted to ≥270 days post treatment (**Figure 4D**).

#### **Figure 3.**

*Partial qPCR characterization of the miRNA expression in the Control- and mPEG-MLR. (A–C) Clustergram (A), Volcano Plot (B) and Log2 Fold (C) analyses of the miRNA expression in the mPEG-MLR and Control-MLR relative to resting cells. (C) Because of the complexity of miRNA regulation of genes, we have consciously chosen to produce a relatively complex miRNA preparations mimicking normal biology in order to achieve maximal biological functionality. Multiple miRNA changes are noted in the hTA1 miRNA compared to either resting cells (green dashed line = 0) or the proinflammatory hIA1 miRNA preparation. Using miRNA expressing a net ΔLog2 Fold change, significantly different "patterns of expression" are noted between the hTA1 and hIA1 miRNA. This pattern of expression, comprising both INCREASED and DECREASED miRNA species is essential for effective immunomodulation of recipient animals. Values derived from a minimum of 3 independent biological replicates. Unpublished data.*

**79**

Treg:Teff ratio.

to Th17<sup>+</sup>

**Figure 4.**

*Modulating the T Lymphocyte Immune Response via Secretome Produced…*

**5. Tolerogenic TA1: immunomodulation of autoimmune disease**

*Differential effects of TA1 and IA1 on the immune system. (A and B): Secretome production (A) of SYN (resting), IA1 (MLR) and TA1 (mPEG-MLR) gave rise to unique immunomodulatory activity (B). While IA1 enhanced proinflammatory subsets and reduced Treg cells, TA1 enhanced Treg while reducing Teff subpopulations. (C) Attesting to the conserved nature of miRNA, murine TA1 and IA1 exerted significant, dose-dependent, immunomodulatory effects on resting human PBMC. The SYN secretome product had no substantive effects on T cell proliferation and differentiation. Data derived from Refs: [32–43] TA1 administration induces a persistent tolerogenic state in immunocompetent mice. (D) As shown, CD4+*

Autoimmune destruction of pancreatic islets gives rise to T1D and occurs via T cell dependent pathways [55–57]. Elucidation of the role of T cells in T1D has been most effectively examined in the nonobese diabetic (NOD) mouse model. In the NOD mouse, evidence suggests that a deficit in Treg control over diabetogenic Teff cells leads to the development of insulitis and disease [56–66]. Indeed, changes in the Treg:Teff ratio (i.e., balance) can be observed as early as 3–4 weeks of age and becomes more pronounced with disease progression (**Figure 5**) [56]. Human studies have similarly demonstrated that T1D Treg exhibit an impaired ability to suppress Teff [67]. Thus, the emergence of an aggressive diabetogenic lymphocyte response in NOD mice, and likely humans, is dependent upon a change in the

*Treg cells remain elevated for ≥270 days following a SINGLE TA1 administration at age 7–8 weeks. In contrast, RNase-treated TA1 (to degrade the miRNA) had no immunomodulatory effect. Results shown are from a* 

*Foxp3+*

As demonstrated in **Figure 5**, the Treg:Teff ratio (defined as the ratio of Foxp3<sup>+</sup>

 T cells) in control (saline treated) NOD mice decreased with disease progression from 103 in nondiabetic 7 week old mice to only 4.7 in diabetic mice at time of sacrifice (15–30 week). Moreover, control NOD mice exhibited a rapid onset of diabetes with 75% (12 of 16) of the mice becoming diabetic by week 19. Subsequent to week 19, no additional mice became diabetic. In contrast, a single dosing (3 injections at 2 days intervals) of the TA1 therapeutic at 7 weeks of age dramatically altered both the incidence and rate of progression of the T1D in the NOD mouse. By week 19 only 13% (2 of 15) of the TA1 treated mice became diabetic with an additional 4 mice becoming diabetic between weeks 21 and 23 (total diabetic 6/15; 40%). Mechanistically, these findings were associated with

*DOI: http://dx.doi.org/10.5772/intechopen.86598*

*minimum of 8 animals per group Unpublished data.*

*Modulating the T Lymphocyte Immune Response via Secretome Produced… DOI: http://dx.doi.org/10.5772/intechopen.86598*

#### **Figure 4.**

*Cells of the Immune System*

PBMC as evidenced via clustergram (**Figure 3A**), volcano plot (**Figure 3B**) and Log2 Fold (**Figure 3C**) miRNA expression analyses. Importantly, as shown in **Figure 3C**, the control- and mPEG-MLRs show unique patterns of expression. While there are some similarities in the pattern of expression there are significant disparity in miRNAs expressed as well (not shown are the miRNA unchanged from resting cells). Importantly, the differences in miRNA expression between the Control- and mPEG-MLR leukocyte yield secretomes that exert dramatically different effects when used to treat resting human PBMC or murine splenocytes. Collection of the secretome produced (**Figure 4A**) during the control and polymer modified allorecognition-based MLR yields a reproducible, acellular, miRNA-rich, material that is stable and can be frozen and thawed with minimal decrement to its activity. As schematically presented (**Figure 4B**), TA1 upregulates regulatory T cell populations (e.g., Treg) while simultaneously downregulating Teff (e.g., Th17 and Th1) cells. In contrast, the proinflammatory IA1 increases Teff while decreasing Treg cells. Of note, the secretome from resting cells (SYN) has minimal to no effect on human or mouse immune cells. Moreover, due to the conserved nature of mammalian miRNA, cross species efficacy is observed with both TA1 and IA1. As shown in **Figure 4C**, murine splenocyte produced TA1 and IA1 exerted dose-dependent effects on a

CD4+

T cell proliferation. Hence, a polymer-based,

T cell proliferation and

human MLR with murine-sourced TA1 reducing CD3+

CD4+

of normal mice that persisted to ≥270 days post treatment (**Figure 4D**).

alloresponse manufacturing system may provide a unique avenue for more effectively, and safely, modulating the Treg:Teff cell ratio via the production of therapeutically effective TA1 and IA1 miRNA-based therapeutics [32–43]. Importantly, the effects of TA1 and IA1 immunotherapy was persistent. In murine studies, a single dosing of TA1 to mice resulted in significant increase in Treg cells within the spleen

*Partial qPCR characterization of the miRNA expression in the Control- and mPEG-MLR. (A–C) Clustergram* 

*(A), Volcano Plot (B) and Log2 Fold (C) analyses of the miRNA expression in the mPEG-MLR and Control-MLR relative to resting cells. (C) Because of the complexity of miRNA regulation of genes, we have consciously chosen to produce a relatively complex miRNA preparations mimicking normal biology in order to achieve maximal biological functionality. Multiple miRNA changes are noted in the hTA1 miRNA compared to either resting cells (green dashed line = 0) or the proinflammatory hIA1 miRNA preparation. Using miRNA expressing a net ΔLog2 Fold change, significantly different "patterns of expression" are noted between the hTA1 and hIA1 miRNA. This pattern of expression, comprising both INCREASED and DECREASED miRNA species is essential for effective immunomodulation of recipient animals. Values derived from a minimum of 3* 

the murine IA1 enhancing CD3+

**78**

*independent biological replicates. Unpublished data.*

**Figure 3.**

*Differential effects of TA1 and IA1 on the immune system. (A and B): Secretome production (A) of SYN (resting), IA1 (MLR) and TA1 (mPEG-MLR) gave rise to unique immunomodulatory activity (B). While IA1 enhanced proinflammatory subsets and reduced Treg cells, TA1 enhanced Treg while reducing Teff subpopulations. (C) Attesting to the conserved nature of miRNA, murine TA1 and IA1 exerted significant, dose-dependent, immunomodulatory effects on resting human PBMC. The SYN secretome product had no substantive effects on T cell proliferation and differentiation. Data derived from Refs: [32–43] TA1 administration induces a persistent tolerogenic state in immunocompetent mice. (D) As shown, CD4+ Foxp3+ Treg cells remain elevated for ≥270 days following a SINGLE TA1 administration at age 7–8 weeks. In contrast, RNase-treated TA1 (to degrade the miRNA) had no immunomodulatory effect. Results shown are from a minimum of 8 animals per group Unpublished data.*

#### **5. Tolerogenic TA1: immunomodulation of autoimmune disease**

Autoimmune destruction of pancreatic islets gives rise to T1D and occurs via T cell dependent pathways [55–57]. Elucidation of the role of T cells in T1D has been most effectively examined in the nonobese diabetic (NOD) mouse model. In the NOD mouse, evidence suggests that a deficit in Treg control over diabetogenic Teff cells leads to the development of insulitis and disease [56–66]. Indeed, changes in the Treg:Teff ratio (i.e., balance) can be observed as early as 3–4 weeks of age and becomes more pronounced with disease progression (**Figure 5**) [56]. Human studies have similarly demonstrated that T1D Treg exhibit an impaired ability to suppress Teff [67]. Thus, the emergence of an aggressive diabetogenic lymphocyte response in NOD mice, and likely humans, is dependent upon a change in the Treg:Teff ratio.

As demonstrated in **Figure 5**, the Treg:Teff ratio (defined as the ratio of Foxp3<sup>+</sup> to Th17<sup>+</sup> T cells) in control (saline treated) NOD mice decreased with disease progression from 103 in nondiabetic 7 week old mice to only 4.7 in diabetic mice at time of sacrifice (15–30 week). Moreover, control NOD mice exhibited a rapid onset of diabetes with 75% (12 of 16) of the mice becoming diabetic by week 19. Subsequent to week 19, no additional mice became diabetic. In contrast, a single dosing (3 injections at 2 days intervals) of the TA1 therapeutic at 7 weeks of age dramatically altered both the incidence and rate of progression of the T1D in the NOD mouse. By week 19 only 13% (2 of 15) of the TA1 treated mice became diabetic with an additional 4 mice becoming diabetic between weeks 21 and 23 (total diabetic 6/15; 40%). Mechanistically, these findings were associated with

a systemic alteration of the immune system as noted in **Figure 5**. In control NOD mice, the progression to diabetes was characterized by significantly elevated levels of most proinflammatory Teff (e.g., INF-γ<sup>+</sup> , Th17<sup>+</sup> , and IL-2<sup>+</sup> ) lymphocytes and a corresponding decrease in regulatory subsets. In contrast, TA1 therapy dramatically and significantly blunted the expansion of Teff cells (as exemplified by INF-γ<sup>+</sup> , Th17<sup>+</sup> , and IL-2<sup>+</sup> lymphocytes; **Figure 5A**) relative to diabetic or nondiabetic control NOD mice coupled with a simultaneous increase in a broad range of tolerogenic/anergic regulatory T cell subsets (e.g., foxp3<sup>+</sup> , IL-10<sup>+</sup> , TGF-β<sup>+</sup> ; **Figure 5B**) in the pancreatic lymph node. These studies also demonstrated that TA1 treated NOD mice had significant numbers of histologically normal pancreatic islets while no normal islets were identified in the untreated mice [40]. It is worth noting that all diabetic mice (control and TA1-treated) exhibited significantly lower levels of these tolerogenic cells than did the 30 week old nondiabetic (control or TA1) mice. Moreover, the effects of TA1-miRNA therapy were not localized to the pancreatic lymph node microenvironment. Analyses of the T cell subsets present in the spleen and brachial lymph node of control and TA1 treated NOD mice (diabetic and nondiabetic) similarly demonstrated dramatic changes in the

#### **Figure 5.**

*The autoimmune disease of T1D process is mediated by a decrease in the Treg:Teff ratio and can be prevented by TA1 administration (top). Treatment with the TA1 miRNA product prevents the decrease and, in fact, significantly increases the Treg:Teff ratio. The increased Treg:Teff ratio is protective as evidenced by the finding that the majority of TA1 treated animals remained normoglycemic. Shown in the blue bars are the Treg:Teff ratio for TA1 treated mice who were diabetic (ratio of 70) and nondiabetic (ratio of 255). Mechanistically, TA1 immunotherapeutic significantly altered the expression of multiple proinflammatory (A) and tolerogenic/ anergic (B) T cell subsets. These changes were systemic in nature as shown by changes in not only the pancreatic lymph node but in other immune tissues (spleen and brachial lymph node). Diabetic tissues were harvested at time of conversion, nondiabetic tissues were harvested at week 30. Diabetic values are the mean ± SD of 12 saline and 6 TA1 treated NOD mice. Nondiabetic results are the mean ± SD of 4 saline and 9 TA1 treated NOD mice. Derived from Ref. [40].*

**81**

*Modulating the T Lymphocyte Immune Response via Secretome Produced…*

Teff cell populations (**Figure 5A**, right) and tolerogenic T cells (**Figure 5B**, right). These findings demonstrate that miRNA-based TA1 therapeutic, directly targets the Treg:Teff ratio yielding a systemic protolerogenic state both *in vivo* (mouse) and *in vitro* (human and mouse) suggesting that this approach would be of utility in a broad range of autoimmune diseases. Furthermore, due to the persistence of the immunomodulatory activity in mice (**Figure 4**), TA1-like drugs could, poten-

tially, dramatically reduce the need for chronic administration of drugs.

**6. Proinflammatory IA1: enhancing the immune response to cancer**

T cells plays a critical role in the anticancer inflammatory responses. An effective anticancer proinflammatory T cell response is dependent upon the activation of Teff cells. Normally, T cells are activated upon ligation of their antigen receptors with specific cognate antigens [68]. However, because of the low frequency of cancer antigen-specific lymphocytes, the immune response to cancers can be initially, and all too often remains, weak. While previous studies have attempted to enhance the anticancer T cell response using pan T cell mitogens (e.g., phytohemagglutinin; PHA), cytokines (e.g., IL-2), or monoclonal antibodies (e.g., anti-CD3 and anti-CD28) the overly robust T cell response arising from these approaches often induced significant systemic toxicity leading to the suspension or abrogation of multiple clinical trials [69–74]. In contrast, in an allorecognition response only 1–10% of T cells are alloreactive [75]. Hence, the IA1 therapeutic, derived from a bioreactor allorecognition response (MLR), is expected to activate endogenous T

To assess IA1's ability to enhance the anticancer activity of resting PBMC, cells were treated for 24 hours with IA1 and overlaid on HeLa and SH-4 cancers cells. Cancer cell proliferation was then followed for 168 hours. Importantly, IA1 exerted no toxicity to resting PBMC but, as shown in **Figure 4**, induced significant

proinflammatory subsets thus decreasing the Teff:Treg ratio. However, as predicted by the biology of the alloresponse, IA1-mediated T cell proliferation was much more restrained than that induced by the anti-CD3/anti-CD28 or PHA stimulation [43]. This finding suggests that the systemic toxicity, relative to pan T cell activators, should be greatly reduced. Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC (**Figure 6**). The anti-proliferation effect of IA1-activated PBMC was noted within ~12 hours vs. 4–5 days for resting cells. These findings demonstrate that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent proinflammatory, anticancer, effect on resting lymphocytes. The potential utility and use of IA1 in Adoptive Cell Therapy (ACT) is diagrammatically shown in **Figure 6**. The bioproduction of IA1 is both inexpensive and rapid (5 days) and the IA1 can be stored for long periods (several months frozen in the laboratory; data not shown). Moreover, neither IA1 or TA1 production actually requires donor specific tissues (PBMC) making these secretome-based therapeutics an "offthe-shelf" immune adjuvant. Most importantly for patient care, *ex vivo* activation of lymphocytes is rapid (24 hours). The rapidity of this approach is in stark contrast to the weeks to months necessary for production and expansion of CAR-T cells. Hence, IA1 activation of autologous PBMC could be employed as a first line therapy or, potentially, be used as an immunotherapeutic bridge while CAR-T cells are produced. Due to the simplicity and low cost of the approach, multiple rounds could be used as necessary with large numbers of autologous PBMC employed. Indeed, due to the ability to infuse large numbers of IA1 treated autologous cells, enhanced recognition

(CD4+

and CD8+

) skewed towards

*DOI: http://dx.doi.org/10.5772/intechopen.86598*

cells in a more controlled manner, with less toxicity.

activation (e.g., proliferation) of resting CD3+

*Modulating the T Lymphocyte Immune Response via Secretome Produced… DOI: http://dx.doi.org/10.5772/intechopen.86598*

*Cells of the Immune System*

fied by INF-γ<sup>+</sup>

levels of most proinflammatory Teff (e.g., INF-γ<sup>+</sup>

, and IL-2<sup>+</sup>

range of tolerogenic/anergic regulatory T cell subsets (e.g., foxp3<sup>+</sup>

, Th17<sup>+</sup>

a systemic alteration of the immune system as noted in **Figure 5**. In control NOD mice, the progression to diabetes was characterized by significantly elevated

and a corresponding decrease in regulatory subsets. In contrast, TA1 therapy dramatically and significantly blunted the expansion of Teff cells (as exempli-

nondiabetic control NOD mice coupled with a simultaneous increase in a broad

**Figure 5B**) in the pancreatic lymph node. These studies also demonstrated that TA1 treated NOD mice had significant numbers of histologically normal pancreatic islets while no normal islets were identified in the untreated mice [40]. It is worth noting that all diabetic mice (control and TA1-treated) exhibited significantly lower levels of these tolerogenic cells than did the 30 week old nondiabetic (control or TA1) mice. Moreover, the effects of TA1-miRNA therapy were not localized to the pancreatic lymph node microenvironment. Analyses of the T cell subsets present in the spleen and brachial lymph node of control and TA1 treated NOD mice (diabetic and nondiabetic) similarly demonstrated dramatic changes in the

*The autoimmune disease of T1D process is mediated by a decrease in the Treg:Teff ratio and can be prevented by TA1 administration (top). Treatment with the TA1 miRNA product prevents the decrease and, in fact, significantly increases the Treg:Teff ratio. The increased Treg:Teff ratio is protective as evidenced by the finding that the majority of TA1 treated animals remained normoglycemic. Shown in the blue bars are the Treg:Teff ratio for TA1 treated mice who were diabetic (ratio of 70) and nondiabetic (ratio of 255). Mechanistically, TA1 immunotherapeutic significantly altered the expression of multiple proinflammatory (A) and tolerogenic/ anergic (B) T cell subsets. These changes were systemic in nature as shown by changes in not only the pancreatic lymph node but in other immune tissues (spleen and brachial lymph node). Diabetic tissues were harvested at time of conversion, nondiabetic tissues were harvested at week 30. Diabetic values are the mean ± SD of 12 saline and 6 TA1 treated NOD mice. Nondiabetic results are the mean ± SD of 4 saline and 9 TA1 treated NOD* 

, Th17<sup>+</sup>

lymphocytes; **Figure 5A**) relative to diabetic or

, and IL-2<sup>+</sup>

) lymphocytes

, TGF-β<sup>+</sup>

;

, IL-10<sup>+</sup>

**80**

*mice. Derived from Ref. [40].*

**Figure 5.**

Teff cell populations (**Figure 5A**, right) and tolerogenic T cells (**Figure 5B**, right). These findings demonstrate that miRNA-based TA1 therapeutic, directly targets the Treg:Teff ratio yielding a systemic protolerogenic state both *in vivo* (mouse) and *in vitro* (human and mouse) suggesting that this approach would be of utility in a broad range of autoimmune diseases. Furthermore, due to the persistence of the immunomodulatory activity in mice (**Figure 4**), TA1-like drugs could, potentially, dramatically reduce the need for chronic administration of drugs.
