*1.2. T Cell Based Therapy*

Elaborate studies in the past 10 years have improved our understanding regarding the adverse effects hyperactive T cells have on endogenous beta cells. These potentially pathogenic migratory cells comprise CD4<sup>+</sup> and CD8<sup>+</sup> T cell subpopulations, B cells, dendritic cells, and macrophages, which have specificity towards islets of Langerhans. The T cells are held in check by various regulatory mechanisms and by a special T cell population known as regulatory T cells (Tregs). An imbalance/defect in this control mechanism and/or dysfunctional Treg population might be one of the causes for the onset of T1D [63]. Various attempts have been made in the direction of understanding and identifying the T cell markers which could mediate the strengthening of immunoregulation [64]. Sorting out the molecular profile of the dysfunctional Treg population and introduction of immunomodulatory agents against these markers may reveal promising targets for T cell immunotherapy [65–68]. The current and developing immunotherapies aim at either preventing the autoimmune response or re-establishing the regulatory control over the autogenic T cell population.

The CD4<sup>+</sup> T cells do not cause beta cell death through direct contact, but rather secrete cytokines to promote recruitment of other immune cells. These inflammatory cytokines, such as IFNγ, IL-1β, and TNFα, also stimulate beta cell death, thereby aggravating islet loss during T1D. On the other hand, CD8<sup>+</sup> T cells lead to beta cell death through direct contact with the beta cells [5,69]. CD4<sup>+</sup> T cells differentiate into a variety of helper T cells, which have their unique cytokine profiles that give them effector functions adapted to a variety of infections [70]. Manipulating these effector or regulatory CD4<sup>+</sup> T cells response is a promising immunotherapy strategy in various autoimmune disorders. Keeping these factors in mind, Eichmann et al. studied the effects of co-stimulation blockade using abatacept over CD4<sup>+</sup> memory T cells and the consequent decline in the beta cell function [71]. Their treatment demonstrated a substantial alteration in the population of CD4<sup>+</sup> cells and Treg cells. Their results also indicated that this approach only affects conventional CD4<sup>+</sup> but not CD8<sup>+</sup> T cell populations. Similarly, Long et al. used Teplizumab to enhance the secretion of inhibitory molecules to reduce the population of CD4<sup>+</sup> and CD8<sup>+</sup> cells, which delayed the onset of T1D [72]. Autoreactive CD8<sup>+</sup> T cells have heterogenous phenotypes and their expression is seen to be affected by the rate of progression of T1D [73]. Elevated expression of activated islet-reactive CD8<sup>+</sup> memory T cells was predominant in T1D patients who demonstrated a rapid loss of C-peptide, while expression of multiple inhibitory markers, limited cytokine levels, and reduced proliferation marked a slower rate of progression of T1D [74].

Identification of markers in correcting the function of dysfunctional Treg cells can also work in the direction of reversal of autoimmune response [75]. These Treg-based therapeutic approaches can be helpful to restore tolerance in the T cell-mediated autoimmune responses [66,76]. Tregs with phenotype CD8+CD25+FOXP3<sup>+</sup> have been seen to effectively suppress the activity of pathogenic T cells and decrease the population of CD8<sup>+</sup> effector T cells [77]. Serr et al. identified HLA-DQ8-restricted insulin-specific CD4<sup>+</sup> T cells and demonstrated efficient human insulin-specific Foxp3<sup>+</sup> Treg-induction after subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo [78]. Functional chimeric antigen receptors (CARs) against insulin in conjunction with FOXP3 can be used to modify naïve effector T cells to specific Treg cells in order to redirect their specificity towards T1D [79]. This approach is expected to result in high specificity, which would minimize the off-target impacts. Modulation and engineering of these Tregs also face drawbacks such as insufficient population, stability of modified expression, and antigen specificity.

More recently, nanomedicine has introduced novel techniques which are significantly capable of altering the immune response [80–82]. This precise control over the immunomodulation by the use of nanoparticles are proficient in inducing immune tolerance, ranging from triggering the pathogenic T cells to Treg cells, and further into effector T cell populations [83]. One of the approaches where nanoparticles have found their use is by using dextran particles to administer autoantigen and immunosuppressant (rapamycin,) which selectively affect the effector T cells without global immunosuppression [84]. It also resulted in a reduction in the proliferation of CD4<sup>+</sup> T cells while an increase was observed in the ratio of FOXP3<sup>+</sup> to IFNγ <sup>+</sup> T cells. These microparticle-based platforms were effective in altering multiple immune cell functions by selectively inhibiting disease-associated T cell immunity and leaving the general immune responses unbroken. Bergot et al. hypothesized whether the tolerizing immunotherapy with a single peptide might be effective to control T1D, which is guided by multiple antigens [85]. They co-encapsulated an autoantigen (chromogranin A, ChgA) along with 1α,25-dihydroxyvitamin D3 in liposomal bilayer and monitored the specific autoimmune response. Liposome administration subcutaneously, but not intravenously, induced ChgA-specific Foxp3<sup>+</sup> and Foxp3<sup>−</sup> PD1<sup>+</sup> CD73<sup>+</sup> ICOS<sup>+</sup> IL-10<sup>+</sup> peripheral regulatory T cells in prediabetic mice, and liposome administration at the onset of hyperglycemia significantly delayed diabetes progression. Their work deduced that the liposomes encapsulated the single CD4<sup>+</sup> peptide, and vitamin D3 analogues induce ChgA-specific CD4<sup>+</sup> T cells that regulate CD4<sup>+</sup> and CD8<sup>+</sup> self-antigen specificities and autoimmune diabetes in NOD mice. On similar lines, Jamison et al. fabricated poly(lactideco-glycolide) (PLG) nanoparticles and loaded with Insulin–ChgA hybrid peptide in order to monitor the balance between effector and regulatory T cells [86]. Administration of hybrid insulin peptide-coupled PLG NPs was found to prevent diabetes by impairing the ability of CD4<sup>+</sup> T cells to produce proinflammatory cytokines through induction of anergy, leading to an increase in the ratio of Foxp3<sup>+</sup> regulatory T cells to IFN-γ + effector T cells. It was also observed that interleukin-2 (IL-2) could enhance the Tregs, which in turn maintained their control over the pathogenic T cells. Aboelnazar et al. studied this relation as a therapeutic strategy and fabricated IL-2-loaded chitosan nanoparticles [87]. They found that low availability of IL-2 in the cellular microenvironment, an inverse correlation between Treg and natural killer (NK) cell expression which was also related to the expression of FOXP3 on Treg cells. IL-6 receptor-mediated signaling also plays a role in development of T cells, which then take part in T1D pathogenesis. Greenbaum et al. attempted to modulate the T cell phenotypes by blocking IL-6 using tocilizumab [88]. They found that while tocilizumab reduced T cell IL-6 signaling, it did not have any effect on CD4<sup>+</sup> T cell phenotypes. No significant difference in the slowing of beta cell loss was observed. Antigen-specific T cell immune tolerance can also be induced by the use oof nanoparticles. A conjugated system of carboxylated polystyrene beads (PSB) with an immunomodulating peptide, HLA-A\*02:01 restricted epitopes, was seen to successfully induce tolerance and suspend the autoimmune cascade in NOD and transgenic humanized mice [89].

These works are suggestive that engineered nanomaterials can conjugate immunomodulators and target desired precise sites of both adaptive and innate immune responses. The size and surface chemistry of these nanomaterials can be tailored according to the

identified target and can be tuned to respond to specific stimuli. Administration of these modified nanoparticles to the T cell family involved in the autoimmune response to T1D can successfully aim to restore immune tolerance and regulatory functions of the immune system. These studies have been tabulated in Table 1 below.


**Table 1.** Summary of strategies for targeting and regulating T cell population and function towards T1D.
