Treatment strategies for PD are currently aimed at symptomatic relief, whereas causative and disease-modifying therapies are not available to date. Although generally not considered a classical neuroinflammatory disease, evidence is mounting that PD patients suffer from an early shift of the immune system towards pro-inflammation and inflammatory T cell responses contributing to neurodegeneration [
6‐
8,
37]. Dysregulation of CD4
+ Treg number and activity has been shown to contribute to the development of the pro-inflammatory condition in PD [
22‐
24]. Here we demonstrate in the hαSyn PD mouse model [
6,
36] that immune modulation through Treg expansion by CD28SA delivery at an early disease stage prevents the development of a pro-inflammatory profile and reduces dopaminergic neurodegeneration in the nigrostriatal tract of hαSyn PD mice.
Treg are pivotal in suppressing the development of unwanted autoimmune responses and are described to be dysregulated in numbers and function in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren’s syndrome among others [
38]. In the MPTP mouse model of PD a neuroprotective and anti-inflammatory effect of CD4
+ Treg has already been shown by either adoptively transferring Treg [
16,
19,
39,
40] or indirect Treg expansion [
17,
18,
28,
29]. Moreover, in PD patients, safety and immunomodulatory effects of sargramostim, a recombinant human GM-CSF that induces Treg via tolerogenic dendritic cells [
41], was demonstrated in a phase 1 trial [
30,
31].
Here, we tested if a direct way to expand and activate Treg by use of a single dose of CD28SA given at an early disease stage reduces neurodegeneration in the hαSyn PD mouse model. This model has already been shown to faithfully recapitulate many pathophysiological hallmarks of human PD including progressive motor deficits, dopaminergic neurodegeneration, Lewy-like brain pathology and neuroinflammation [
6,
36]. Whether immune modulation can reduce neurodegeneration in this PD model was not demonstrated so far. We found an elevated percentage of CD4
+CD25
+FoxP3
+ Treg among CD4
+ T cells in peripheral lymphoid organs of hαSyn PD mice already 10 days after disease induction by hαSyn vector injection. This observation indicates a Treg dysregulation in PD mice that is in line with human PD data on disease-related alteration of Treg numbers [
22‐
24]. Of note, the amino acid sequence of the delivered pathologic human A53T-αSyn differs from physiologic mouse αSyn in six amino acids only, thereby putatively acting in parts as a self-antigen and driving the murine self-antigen-specific Treg expansion by the CD28SA to counteract the pro-inflammatory immune response. In line with this observation, fibrillar αSyn was demonstrated to increase the percentage of CD3
+CD4
+FoxP3
+ Treg after subcutaneous inoculation in mice, thereby suggesting that α-synuclein might have a role in controlling Treg generation or expansion [
42].
The observed early Treg expansion and elevation of IL-10 levels in cervical lymph nodes and spleen of hαSyn PD and EV mice, 3 days after CD28SA delivery, was in agreement with reports from CD28SA treatment in healthy mice and in models for neuroinflammatory diseases such as glucose-6-phosphate isomerase (G6PI)-induced arthritis as model for rheumatoid arthritis and experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis [
34,
43,
44]. These data show that Treg of hαSyn PD mice are susceptible to CD28SA treatment despite of the observed dysregulation. In contrast to the pronounced Treg response in peripheral lymphatic organs to CD28SA administration, Treg number in brain was increased only in hαSyn PD mice while CD28SA-treated EV and PBS-injected control groups revealed lower Treg numbers. This is indicative of a migration and a reactivation of Treg in hαSyn PD mouse brains after peripheral priming and activation in cervical lymph nodes by hαSyn proteins that have drained from the brain via lymphatic vessels as previously suggested for this hαSyn PD mouse model [
6]. In addition, these data demonstrate that CD28SA acts in the peripheral immune compartment and does not need to penetrate the blood–brain barrier to access the brain to have an impact. Importantly, it was shown in a tumor implantation model that blood–brain barrier integrity is already reestablished 7 days after intracerebral surgery, assessed by
3H-mannitol and Evan’s Blue permeability [
45], thereby excluding a blood–brain barrier leakage as cause for the increased Treg number in brain of hαSyn PD mice at this early disease stage.
Administration of a single dose of CD28SA at an early disease stage prevented loss of dopaminergic perikarya in the SN and, strikingly, also ameliorated dopaminergic terminal loss in the striatum of hαSyn PD mice over controls that was accompanied by functional motor recovery on the rotarod test. In addition, less neuroinflammation was found in CD28SA-treated hαSyn PD mice compared to PBS controls with decreased numbers of CD4
+ and CD8
+ T cells in the nigrostriatal tract, reduced percentage of CD69
+-activated brain T cells among the CD4
+ and CD8
+ population and normalization of elevated IL-2 levels. Interestingly, aggregated α-synuclein pathology, assessed after proteinase K-digestion in hαSyn PD mice did not change significantly after CD28SA-treatment. In line with this finding, in a microglia-specific α-synuclein-overexpression mouse model dopaminergic neurons were observed to degenerate independent of their intraneuronal pathological α-synuclein load [
46]. Alleviation of behavioral deficits and attenuation of inflammation by CD28SA-treatment has been observed in various rodent disease models, such as for rheumatoid arthritis and multiple sclerosis and stroke [
34,
35,
44]. Moreover, a neuroprotective effect of CD28SA-treatment was shown for models of ischemic stroke resulting in reduction of infarct size [
35]. A neuroprotective effect on dopaminergic neurons in the SN by genetic ablation of T cells has been demonstrated in the hαSyn PD mouse model [
6]. In addition, experimental deep brain stimulation of the subthalamic nucleus was found to rescue dopaminergic SN perikarya in the equivalent hαSyn PD rat model [
47,
48]. However, in both studies dopaminergic terminals and axons still degenerated in the respective hαSyn PD rodents despite the rescue of dopaminergic perikarya. This indicates that CD28SA treatment in hαSyn PD mice has an additional protective effect on axonal degeneration. Of note, a delayed CD28SA therapy at 4 weeks after AAV injection, when ~ 15–20% of SN neurons are already degenerated, did not prevent progressive dopaminergic neurodegeneration, thereby underlining the disease-modifying effect of an early CD28SA treatment. To analyze whether neuroprotection in hαSyn PD mice was mediated by Treg or rather another (direct) CD28SA effect, we adoptively transferred expanded Treg into hαSyn PD mice 1 week after disease induction and observed a rescue of dopaminergic perikarya and terminals compared to hαSyn control mice at 10 weeks. CD4
+ and CD8
+ T cell numbers were reduced in SN and striatum of hαSyn PD mice that received Treg. These data underline that the neuroprotective effect of CD28SA-treatment is indeed mediated by Treg. To assess the mechanism of action of Treg expansion in hαSyn PD mice, we analyzed CD8
+ brain T cell activation with the CD69 marker three days after CD28SA delivery and observed a slight suppression of CD8
+CD69
+ T cells in the brain T cells in hαSyn PD mice and, more strikingly, a suppression of early IL-2 expression in cervical lymph nodes and spleen of PD mice of ~ 30–45% three days after CD28SA injection. These data demonstrate that CD28SA has an early anti-inflammatory effect, especially on the peripheral immune compartment of hαSyn PD mice. It is likely that peripheral, CD28SA-expanded Treg control Teff responses directly or indirectly through dendritic cells, leading to reduced Teff activation and infiltration into the CNS and consecutively less microglia activation. This hypothesis is supported by the observation of an early and strong upregulation of IL-2 from T cells in cervical lymph nodes of hαSyn PD mice [
6]. As IL-2 is described to promote differentiation of naïve T cells into effector T cells upon antigen-dependent priming [
49], hαSyn-specific memory T cells can then infiltrate the brain and become reactivated upon encountering their cognate antigen (α-synuclein-specific peptides) on neurons [
50,
51]. Subsequently, activated microglia can then induce neurodegeneration to dopaminergic neurons [
46,
52]. In contrast, using the general astrocyte marker GFAP, we did not observe any significant alteration of astrocytes in hαSyn PD mice. In a transgenic hA53T-α-synuclein PD mouse model, activated microglia converted astrocytes to neurotoxic A1 astrocytes [
53]. Therefore, future studies are necessary to assess the impact of reactive astrocytes subtypes (A1- and A2-specific) in this hαSyn PD model. Although clinical trials have been undertaken to translate CD28SA into human patients [
54], this step remains uncertain. Independent of the method, our data indicate that protocols for Treg expansion in humans may be considered for PD patients.