Investigating viral and autoimmune T cell responses associated with post-acute sequelae of COVID-19.

Post-acute sequelae of COVID-19 (PASC), or Long COVID, is a chronic condition following acute SARS-CoV-2 infection. Symptoms include exertion fatigue, respiratory issues, myalgia, and neurological manifestations such as 'brain fog,' posing concern for their debilitating nature and potential role in other neurological disorders. However, the underlying potential pathogenic mechanisms of the neurological complications of PASC is largely unknown. Herein, we investigated differences in antigen-specific T cell responses from the peripheral blood towards SARS-CoV-2, latent viruses, or neuronal antigens in 14 PASC individuals with neurological manifestations (PASC-N) versus 22 individuals fully recovered from COVID-19. We employed Activation Induced Marker (AIM), ICS and FluoroSpot assays to determine the specificity and magnitude of CD4+ and CD8+ T cell responses towards SARS-CoV-2 (Spike and rest of proteome), latent viruses (CMV, EBV), and several neuronal antigens. Overall, we observed similar antigen-specific T cell frequencies and cytokine effector T cell responses between PASC donors compared to recovered controls for all antigens tested (viral or autoantigen) in both CD4+ and CD8+ T cell compartments. Our findings suggest that PASC-N does not appear to be associated with changes in antigen-specific T cell responses towards a subset of disease-relevant targets, but more studies in a larger cohort are needed to confirm these unaltered responses.

PINK1 is a target of T cell responses in Parkinson's disease.

Parkinson's disease (PD) is associated with autoimmune T cells that recognize the protein alpha-synuclein in a subset of individuals. Multiple neuroantigens are targets of autoinflammatory T cells in classical central nervous system autoimmune diseases such as multiple sclerosis (MS). Here, we explored whether additional autoantigenic targets of T cells in PD. We generated 15-mer peptide pools spanning several PD-related proteins implicated in PD pathology, including GBA, SOD1, PINK1, parkin, OGDH, and LRRK2. Cytokine production (IFNγ, IL-5, IL-10) against these proteins was measured using a fluorospot assay and PBMCs from patients with PD and age-matched healthy controls. This approach identified unique epitopes and their HLA restriction from the mitochondrial-associated protein PINK1, a regulator of mitochondrial stability, as an autoantigen targeted by T cells. The T cell reactivity was predominantly found in male patients with PD, which may contribute to the heterogeneity of PD. Identifying and characterizing PINK1 and other autoinflammatory targets may lead to antigen-specific diagnostics, progression markers, and/or novel therapeutic strategies for PD.

Unaltered T cell responses to common antigens in individuals with Parkinson's disease.

BACKGROUND AND OBJECTIVES: Parkinson's disease (PD) is associated with a heightened inflammatory state, including activated T cells. However, it is unclear whether these PD T cell responses are antigen specific or more indicative of generalized hyperresponsiveness. Our objective was to measure and compare antigen-specific T cell responses directed towards antigens derived from commonly encountered human pathogens/vaccines in patients with PD and age-matched healthy controls (HC). METHODS: Peripheral blood mononuclear cells (PBMCs) from 20 PD patients and 19 age-matched HCs were screened. Antigen specific T cell responses were measured by flow cytometry using a combination of the activation induced marker (AIM) assay and intracellular cytokine staining. RESULTS: Here we show that both PD patients and HCs show similar T cell activation levels to several antigens derived from commonly encountered human pathogens/vaccines in the general population. Similarly, we also observed no difference between HC and PD in the levels of CD4 and CD8 T cell derived cytokines produced in response to any of the common antigens tested. These antigens encompassed both viral (coronavirus, rhinovirus, respiratory syncytial virus, influenza, cytomegalovirus) and bacterial (pertussis, tetanus) targets. CONCLUSIONS: These results suggest the T cell dysfunction observed in PD may not extend itself to abnormal responses to commonly encountered or vaccine-target antigens. Our study supports the notion that the targets of inflammatory T cell responses in PD may be more directed towards autoantigens like α-synuclein (α-syn) rather than common foreign antigens.

Central and Peripheral Inflammation: Connecting the Immune Responses of Parkinson's Disease.

Inflammation has increasingly become a focus of study in regards to Parkinson's disease (PD). Moreover, both central and peripheral sources of inflammation have been implicated in the pathogenesis of PD. Central inflammation consisting of activated microglia, astroglia, and T cell responses within the PD central nervous system; and peripheral inflammation referring to activated innate cells and T cell signaling in the enteric nervous system, gastrointestinal tract, and blood. This review will highlight important work that further implicates central and peripheral inflammation in playing a role in PD. We also discuss how these two distant inflammations appear related and how that may be mediated by autoantigenic responses to α-syn.

Profiling Human Cytomegalovirus-Specific T Cell Responses Reveals Novel Immunogenic Open Reading Frames.

Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel open reading frames (ORFs) is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes ∼150 previously described and ∼200 recently described potential novel ORFs by using an ex vivo gamma interferon (IFN-γ) FluoroSpot assay. We identified 235 unique HCMV-specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. IMPORTANCE To understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV "ORFeome" to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of the anti-CMV T cell response and providing new targets for vaccine design.

CD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson's disease.

α-Synuclein, a key pathological component of Parkinson's disease, has been implicated in the activation of the innate and adaptive immune system. This immune activation includes microgliosis, increased inflammatory cytokines, and the infiltration of T cells into the CNS. More recently, peripherally circulating CD4 and CD8 T cells derived from individuals with Parkinson's disease have been shown to produce Th1/Th2 cytokines in response to α-synuclein, suggesting there may be a chronic memory T cell response present in Parkinson's disease. To understand the potential effects of these α-syn associated T cell responses we used an α-synuclein overexpression mouse model, T cell-deficient mice, and a combination of immunohistochemistry and flow cytometry. In this study, we found that α-synuclein overexpression in the midbrain of mice leads to the upregulation of the major histocompatibility complex II (MHCII) protein on CNS myeloid cells as well as the infiltration of IFNγ producing CD4 and CD8 T cells into the CNS. Interestingly, genetic deletion of TCRß or CD4, as well as the use of the immunosuppressive drug fingolimod, were able to reduce the CNS myeloid MHCII response to α-synuclein. Furthermore, we observed that CD4-deficient mice were protected from the dopaminergic cell loss observed due to α-syn overexpression. These results suggest that T cell responses associated with α-synuclein pathology may be damaging to key areas of the CNS in Parkinson's disease and that targeting these T cell responses could be an avenue for disease modifying treatments.

Innate and adaptive immune responses in Parkinson's disease.

Parkinson's disease (PD) has classically been defined as a movement disorder, in which motor symptoms are explained by the aggregation of alpha-synuclein (α-syn) and subsequent death of dopaminergic neurons of the substantia nigra pars compacta (SNpc). More recently, the multisystem effects of the disease have been investigated, with the immune system being implicated in a number of these processes in the brain, the blood, and the gut. In this review, we highlight the dysfunctional immune system found in both human PD and animal models of the disease, and discuss how genetic risk factors and risk modifiers are associated with pro-inflammatory immune responses. Finally, we emphasize evidence that the immune response drives the pathogenesis and progression of PD, and discuss key questions that remain to be investigated in order to identify immunomodulatory therapies in PD.

T cell infiltration in both human multiple system atrophy and a novel mouse model of the disease.

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by abnormal accumulation of alpha-synuclein (α-syn) in oligodendrocytes accompanied by inflammation, demyelination, and subsequent synapse and neuronal loss. Little is known about the mechanisms of neurodegeneration in MSA. However, recent work has highlighted the important role of the immune system to the pathophysiology of other synuclein-related diseases such as Parkinson's disease. In this study, we investigated postmortem brain tissue from MSA patients and control subjects for evidence of immune activation in the brain. We found a significant increase of HLA-DR+ microglia in the putamen and substantia nigra of MSA patient tissue compared to controls, as well as significant increases in CD3+, CD4+, and CD8+ T cells in these same brain regions. To model MSA in vivo, we utilized a viral vector that selectively overexpresses α-syn in oligodendrocytes (Olig001-SYN) with > 95% tropism in the dorsal striatum of mice, resulting in demyelination and neuroinflammation similar to that observed in human MSA. Oligodendrocyte transduction with this vector resulted in a robust inflammatory response, which included increased MHCII expression on central nervous system (CNS) resident microglia, and infiltration of pro-inflammatory monocytes into the CNS. We also observed robust infiltration of CD4 T cells into the CNS and antigen-experienced CD4 T cells in the draining cervical lymph nodes. Importantly, genetic deletion of TCR-ß or CD4 T cells attenuated α-syn-induced inflammation and demyelination in vivo. These results suggest that T cell priming and infiltration into the CNS are key mechanisms of disease pathogenesis in MSA, and therapeutics targeting T cells may be disease modifying.

Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD. METHODS: In vitro, we utilized microglia cultures from WT or CIITA -/- mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA -/- mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration. RESULTS: Using ICC and DQ-ovalbumin, we show that CIITA -/- microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration. CONCLUSION: Our data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD.

Peripheral monocyte entry is required for alpha-Synuclein induced inflammation and Neurodegeneration in a model of Parkinson disease.

Accumulation of alpha-synuclein (α-syn) in the central nervous system (CNS) is a core feature of Parkinson disease (PD) that leads to activation of the innate immune system, production of inflammatory cytokines and chemokines, and subsequent neurodegeneration. Here, we used heterozygous reporter knock-in mice in which the first exons of the fractalkine receptor (CX3CR1) and of the C-C chemokine receptor type 2 (CCR2) are replaced with fluorescent reporters to study the role of resident microglia (CX3CR1+) and infiltrating peripheral monocytes (CCR2+), respectively, in the CNS. We used an α-syn mouse model induced by viral over-expression of α-syn. We find that in vivo, expression of full-length human α-syn induces robust infiltration of pro-inflammatory CCR2+ peripheral monocytes into the substantia nigra. Genetic deletion of CCR2 prevents α-syn induced monocyte entry, attenuates MHCII expression and blocks the subsequent degeneration of dopaminergic neurons. These results demonstrate that extravasation of pro-inflammatory peripheral monocytes into the CNS plays a key role in neurodegeneration in this model of PD synucleinopathy, and suggest that peripheral monocytes may be a target of neuroprotective therapies for human PD.