Early biomarker for Parkinson's disease

Abstract

The present invention provides a non-invasive ex vivo method for diagnosis, prognosis or monitoring of Parkinson's disease (PD) in a subject comprising determining the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM) in a biological sample from the subject, as well as a computer program product for implementing the method. Also provided herein are kits (particularly kits for diagnosis, prognosis or monitoring of PD) and uses thereof, the kits comprising: (a) means specifically suitable for determining the ratio of CD8 TEMRA to CD8 TCM; and (b) optionally a reference of said ratio or means for establishing said reference wherein said reference represents a known diagnosis or prognosis of PD wherein (a) and optionally (b) are the only biological agents present in said kit.

Description

Technical Field

[0001] The present invention relates to novel biomarkers and combinations of biomarkers for the early diagnosis or prognosis of Parkinson's disease in subjects, as well as kits for use therein. Background of the Invention

[0002] Parkinson's disease (PD) is a progressive disorder that affects the nervous system and parts of the body controlled by nerves. PD is the second most common neurodegenerative disease after Alzheimer's disease (AD), affecting approximately 10 million people worldwide. Currently, PD is typically diagnosed by a neurologist based on medical history, symptoms, and neurological and physical examinations. Symptoms of PD include tremor, bradykinesia, muscle rigidity, postural and balance impairments, loss of voluntary movement, speech changes, and writing difficulties. The onset of PD symptoms is relatively slow. The first symptom may be a barely perceptible tremor in one hand. Therefore, accurate diagnosis of PD, especially in its early stages, can be challenging. PD leads to a high rate of disability and requires care.

[0003] Several molecular markers derived from cerebrospinal fluid (CSF) have been found to be useful in the diagnosis of Parkinson's disease (PD). However, CSF sampling may cause further discomfort to PD patients. Furthermore, blood α-synuclein species and neurofilament light chains (NfL) have been identified as potentially promising non-invasive biomarkers. However, further research and confirmation in larger cohorts are still needed due to the small sample sizes used in studies identifying these biomarkers. Additionally, while significant differences in blood NfL were observed between PD and atypical Parkinson's disease (APD), no significant differences were found in blood NfL between PD patients and HC patients.

[0004] Since most PD cases cannot be associated with a genetic origin, there is a great need to identify novel, readily available, and validated early window cellular or molecular biomarkers that can help diagnose or prognose idiopathic PD. Overview

[0005] The inventors of this application have unexpectedly discovered that the relationship between the amount of CD8 terminally differentiated effector T cells (CD8 TEMRA) and the amount of CD8 central memory T cells (CD8 TCM) in biological samples from subjects (e.g., expressed as a ratio of CD8 TEMRA to CD8 TCM (CD8 TEMRA:CD8 TCM) or a ratio of CD8 TCM to CD8 TEMRA (CD8 TCM:CD8 TEMRA)) can be used as a highly accurate (e.g., AUC value of at least 0.85, preferably at least 0.9, more preferably at least 0.94, e.g., at least 0.95), readily available, and non-invasive cellular index biomarker for the early diagnosis or prognosis of PD (e.g., within 5 years of disease onset, such as within 5 years of the first sign of motor symptoms). Diagnosis of PD at an early stage of the disease also allows for earlier initiation of treatment, which is crucial for irreversible neurodegenerative diseases like PD. In addition, since the CD8 TEMRA to CD8 TCM ratio allows for very accurate diagnosis or prognosis of PD (preferably early to mid-stage PD), there is no need to measure additional biomarkers, and thus overfitting (which usually occurs when combining a large number of biomarkers) can be avoided.

[0006] Therefore, a first aspect provides a method (e.g., in vitro or ex vivo method) for diagnosing, prognosing, and / or monitoring Parkinson's disease (PD) in a subject, the method comprising determining the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM) (i.e., CD8 TEMRA:CD8 TCM) in a biological sample from the subject. Those skilled in the art will understand that, alternatively, the ratio of CD8 TCM to CD8 TEMRA (i.e., CD8 TCM:CD8 TEMRA) can be used in the methods taught herein. In a particular embodiment, the method further includes the step of determining the quantities of CD8 TEMRA and CD8 TCM in the biological sample from the subject prior to determining the CD8 TEMRA:CD8 TCM ratio. In a particular embodiment, the biological sample is a body fluid sample, preferably a peripheral whole blood sample.

[0007] In a particular implementation, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0008] In a particular implementation, the method includes the following steps:

[0009] (a) Determine the ratio of CD8 TEMRA to CD8 TCM in biological samples from the subjects;

[0010] (b) Compare the CD8 TEMRA to CD8 TCM ratio determined in (a) to a reference value representing a value that allows for the diagnosis or prognosis of PD; and

[0011] (c) Diagnose, prognose, or monitor the PD of the subject based on the comparison in step (b).

[0012] In a particular implementation, the method includes the following steps:

[0013] (i) Determine the ratio of CD8 TEMRA to CD8 TCM in biological samples from the subjects;

[0014] (ii) Compare the CD8 TEMRA to CD8 TCM ratio determined in (i) to a reference value representing a value that allows for the diagnosis or prognosis of PD; and

[0015] (iii) The ratio of CD8 TEMRA to CD8 TCM determined in (i) is found to be different from or not different from the reference value; and

[0016] (iv) Attributing the findings, whether different or not, to a specific diagnosis or prognosis of the subject's PD.

[0017] In a specific embodiment of the method of the present invention, the method includes diagnosing or prognosing PD in the subject based on a determined CD8 TEMRA to CD8 TCM ratio. In a specific embodiment, a significant increase in the CD8 TEMRA to CD8 TCM ratio compared to the reference, when the reference corresponds to a CD8 TEMRA to CD8 TCM ratio in a biological sample unaffected by PD (e.g., a healthy sample), allows for the diagnosis or prognosis of PD in the subject; or

[0018] When the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample affected by PD, the lack of significant difference in the ratio of CD8 TEMRA to CD8 TCM compared to the reference allows for the diagnosis or prognosis of PD in the subject.

[0019] On the other hand, kits are provided (particularly kits for the diagnosis, prognosis, or monitoring of PD), said kits comprising:

[0020] (a) A tool specifically designed for determining the ratio of CD8 TEMRA to CD8 TCM; and

[0021] (b) Optionally, a reference for the ratio or a tool for establishing the reference, preferably wherein the reference represents a known diagnosis or prognosis of PD.

[0022] (a) and (b) are the only biological reagents present in the kit.

[0023] In a particular implementation, the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample unaffected by PD (e.g., a healthy sample), or the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample affected by PD.

[0024] In a particular implementation, the tool specifically designed for determining the ratio of CD8 TEMRA to CD8 TCM includes one or more binding agents that allow CD8 TEMRA and CD8 TCM to be distinguished from other CD8+ T cells.

[0025] Another related aspect provides the use of the kits described herein for the diagnosis, prognosis, or monitoring of PD based on the determination of the ratio of CD8 TEMRA to CD8 TCM in a subject's biological sample.

[0026] Another related aspect provides a computer program product for use in conjunction with a computer having a processor and memory connected to the processor, the computer program product including a computer-readable storage medium thereon having a computer program mechanism encoded thereon, wherein the computer program mechanism causes the computer to perform the methods taught herein.

[0027] Another related aspect provides a computer-executed method for diagnosing, prognosing, or monitoring Parkinson's disease (PD) in a subject, the method comprising determining the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM) in a biological sample from the subject.

[0028] These and other aspects of the invention, as well as preferred embodiments, are described in the following sections and the appended claims. The subject matter of the appended claims is specifically incorporated herein by reference. Brief description of the attached diagram

[0029] Figure 1 Single-cell CyTOF analysis revealed stronger cytotoxicity in early to mid-stage iPD and an immune profile of late differentiation. (A) A graphical representation illustrates the cohort and experimental setup. CyTOF: mass cytometry; CMV: cytomegalovirus; PBMCs: peripheral blood mononuclear cells; HC: healthy controls, n=24; PD: Parkinson's disease patients, n=28.

[0030] Figure 2Early to mid-stage iPD exhibited an increased effector profile in CD8 T cells. (A) The PCA plot shows a unique immunofingerprint based on T cell combinatorial characteristics analyzed using flow cytometry (FCM). (B) The volcano plot shows the most significant (p<0.05, fold change >1.4) decreases (solid inverted pyramids) and increases (hollow triangles) in PD compared to HC. TCM: Central memory type. The dashed line on the y-axis corresponds to a value of 1.3 (p=0.05), while the two dashed lines on the x-axis correspond to log2 values ​​of -0.485 or 0.485 (fold change 1.4). (C) The scatter plot (left) and representative FCM plot (right) show an increase in CD8 TEMRA (CD45O-CD45RA+CCR7-, a simplified gating strategy for TEMRA without considering CD27) in all participants (left), female participants (middle), or male participants (right). TEMRA gating is highlighted as a dashed rectangle in the FCM plot. The combination of markers used to define TEMRA is described in the heading of the y-axis. (D) The scatter plot shows the ratio between CD8TEMRA and CD8 TCM. Results in BD were analyzed using an unpaired two-tailed Student's t-test, without correction for multiple comparisons. Data are presented as mean ± standard deviation (sd). Each symbol represents a measurement (CD) from one individual participant. ns or unlabeled, not significant; *p<=0.05, **p<=0.01, and ***p<=0.001. HC: healthy controls, n=24; PD: Parkinson's disease patients, n=28; FCM, flow cytometry. For C and D, female HC, n=10; female PD, n=8; male HC, n=14; male PD, n=19. Notably, one female PD sample was excluded because the same patient was visited twice within a short period.

[0031] Figure 3 CD8 TEMRA alone, and the ratio of CD8 TEMRA to TCM, are reliable peripherally accessible cellular biomarkers for early to mid-stage iPD. Figures (A, B) show the levels of CD8 TEMRA (CD45RO) in total CD8 T cells. - CCR7 - CD27 - The correlation between the frequency of PD and the duration of disease from the onset of initial symptoms (A) or the duration of disease from clinical diagnosis (B) was calculated. Correlation coefficients and p-values ​​were calculated based on Spearman correlation. (C, D) were based on the CD45RA levels in total CD8 T cells relative to all HC (n=24) in all early to mid-stage PD (C) or patients diagnosed within 5 years (diagnosis <= 5 years, n=11) (D), respectively. + CD45RO- CCR7 - ROC analysis of the frequencies. (E) The figure shows the frequency of CD8 TEMRA (CD45RA) relative to CD8 Treg for each individual participant. + CD45RO - CCR7 - The frequency of CD8 TEMRA (CD45RA) as measured by FCM is shown in Figure (F). The figure represents the frequency of ILC2 as measured by CyTOF for each individual participant. + CD45RO - CCR7 - The (G) diagram shows the frequency of each individual relative to CD8 TEMRA (CD45RO). - CCR7 - CD27 - The ratio between CD8 (CD45RA) and CD8 Treg is used to determine the ratio of CD8 (CD45RA) to CD8 Treg. + CD45RO - CCR7 - The frequency of CD8 TEMRA (CD45RA) is shown in the dashed circles, highlighting the PD-dominated regions. (H) Based on the total CD8 T cells from female PD (n=8) or HC (n=10) alone. + CD45RO - CCR7 - ROC analysis of the frequency of ) for all iPD (n=28) relative to HC (n=24) (I) or female-only iPD (n=8) and HC (n=10) (J), based on CD8 TEMRA (CD45RO) in total CD8 T cells. - CCR7 - CD27 - ROC analysis of the ratio between the frequency of TEMRA (CD45RA) and the frequency of CD8 Tregs. (K, L) were respectively for all iPD (n=28) (K) or patients diagnosed within 5 years (diagnosis <= 5 years, n=11) (L) relative to all HC (n=24), based on TEMRA (CD45RA) in total CD8 T cells. + CD45RO - CCR7 - ) frequency and TCM (CD45RO) + CCR7 + CD27 +ROC analysis of the ratio between the frequencies of TEMRA and TCM in total CD8 T cells based on female (M) iPD (n=8) and HC (n=10) or male (N) iPD (n=19) and HC (n=14) total CD8 T cells. For LN, the corresponding scatter plots are shown in the right subplot and statistical analysis was performed using an unpaired two-sided Student's t-test. Each symbol represents a measurement from one individual participant. Unless otherwise specified, all PD (n=28) and HC (n=24) were used in the analysis. The label "9" is used to highlight the same outlier "PD9" in several plots, which cannot even be based on such outliers. Figure 2 T-cell characteristics in all analyses shown in Figure A were distinguished from HC. The p-values ​​shown for each ROC analysis were tested using a two-tailed test (GraphpadPrism v9.0) to confirm the null hypothesis that the area under the curve (AUC) was indeed equal to 0.50. ROC: Receiver operating characteristic; AUC: Area under the curve; HC: Healthy controls; PD: Parkinson's disease patients. Notably, one female PD sample was excluded because the same patient was accessed twice within a short period. As discussed in this paper, slightly different combinations of markers (CD45RA) were used when using different staining plates. + CD45RO - CCR7 - Or CD45RO - CCR7 - CD27 - ) to define CD8 TEMRA in different subgraphs.

[0032] Figure 4The enhanced expression of the CD8 TEMRA component and the increased co-expression of CD8 cytotoxic molecules were validated in another subcohort. (A) A scatter plot shows the frequency of total CD8 T cells in singles of CD3 or total viable lymphocytes (11 iPDs vs. 12 HCs) analyzed using FCM in cryopreserved PBMCs from another subcohort of the Luxembourg Parkinson's study. (B, C) Scatter plots show the frequency of CD8 TEMRA (B) or TCM (C) in total CD8 T cells defined using different markers. Notably, one sample was excluded from the PDs because it was identified as an outlier by the default settings of the ROUT method in Graphpad. (D) A scatter plot shows the ratio between CD8 TEMRA and CD8 TCM defined using different markers. Results in AD were analyzed using a common one-way ANOVA with a two-stage linear ascending procedure corrected for Benjamini / Krieger / Yekutieli. q-values ​​(FDR) are shown. The median for each group is labeled. Each symbol represents a measurement from an individual participant (ns or unlabeled, non-significant; *q<=0.05, **q<=0.01, and ***q<=0.001). HC: healthy controls, n=12, including 5 men; PD: patients with Parkinson's disease, n=11, including 8 men; FCM: flow cytometry.

[0033] Figure 5 Early to mid-stage iPD showed fewer memory CD8 T cells and no signs of accelerated exhaustion. (A) Scatter plot showing total CD3 in PD and HC as analyzed by FCM. + CD4 + and CD8 +T cell frequency. The parental gated target of CD3+ cells is a live lymphocyte single-cell population. (B) Scatter plot shows the frequency of effector CD8 T cells in total CD8 T cells. (C) Bar plot shows the relative mean proportions of CD45RA vs. CD45RO (left panel) and CCR7 vs. CD45RO tetrads (right panel) of CD8 T cells. (DG) Scatter plot shows the frequency of TCM (central memory type) (D), TM (transitional memory type) (E), naive type (F), and long-lived memory type (G) in CD8 T cells of PD and HC participants (left), female participants (middle), or male participants (right). The combination of markers used to define the corresponding subtypes is described directly in the y-axis headings. Results were analyzed using an unpaired two-tailed Student's t-test. Data are expressed as mean ± standard deviation (SD) for a given group. Each symbol represents a measurement from an individual participant (A, B, DG). ns: Not significant; *p<=0.05, **p<=0.01 and ***p<=0.001. HC: Healthy controls, n=24; PD: Parkinson's disease patients, n=28. Female HC, n=10; Female PD, n=8; Male HC, n=14; Male PD, n=19. Notably, for Figure D, one female PD sample was excluded because the same patient was visited twice within a short period. Description of the implementation plan

[0034] As used herein, the singular forms “a”, “an”, and “the” include the singular and plural objects referred to, unless the context clearly specifies otherwise.

[0035] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” “containing,” and “contains,” and are inclusive or open-ended, and do not exclude other, unlisted members, elements, or method steps. These terms also include “consisting of” and “consisting essentially of,” which have their generally accepted meanings in patent terminology.

[0036] Enumerating numerical ranges by endpoints includes all numbers and fractions within the corresponding range, along with the listed endpoints. This applies to numerical ranges regardless of whether they are introduced by expressing "from...to..." or "...to..." or another expression.

[0037] When referring to measurable values ​​(e.g., parameters, quantities, durations, etc.), the terms “about” or “approximately” as used herein mean encompassing variations in the specified value and variations derived from the specified value, such as variations in the specified value and from the specified value by + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less, provided that such variations are suitable for implementation in the disclosed invention. It should be understood that the values ​​referred to by the modifier “about” or “approximately” are themselves specific and preferably disclosed.

[0038] While the terms “one or more” or “at least one” (e.g., at least one member of one or more members or a group of members) are self-evident, by further example, the term specifically covers references to any one of the members or any two or more of the members, such as, for example, any ≥3, ≥4, ≥5, ≥6, or ≥7 of the members, and up to all of the members. In another example, “one or more” or “at least one” could refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0039] This document includes a discussion of the background of the invention to explain the context of the invention. This should not be construed as an admission that any material mentioned was publicly available, known, or part of common general knowledge in any country at the priority date referred to in any claim.

[0040] Throughout this disclosure, various publications, patents, and published patent specifications are cited by way of identifiable reference. All documents referenced in this specification are incorporated herein by reference in their entirety. In particular, the teachings or sections of such documents specifically mentioned herein are incorporated herein by reference.

[0041] Unless otherwise defined, all terms used in this invention (including technical and scientific terms) have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For further guidance, terminology definitions are included herein to better understand the teachings of this invention. When a particular term is defined in connection with a specific aspect or embodiment of the invention, such connotation or meaning is intended to be applied throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0042] In the following paragraphs, different aspects or embodiments of the invention are defined in more detail. Unless expressly stated to the contrary, each aspect or embodiment so defined may be combined with any other aspect or embodiment. In particular, any feature indicated as preferred or advantageous may be combined with one or more other features indicated as preferred or advantageous.

[0043] Throughout this specification, the references to "an embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, the particular feature, structure, or characteristic can be combined in any suitable manner, as will be apparent to those skilled in the art. Moreover, as those skilled in the art will understand, while some embodiments described herein include certain features but not others included in other embodiments, combinations of features from different embodiments are intended to fall within the scope of the invention and form different embodiments. For example, in the appended claims, any claimed embodiment can be used in any combination.

[0044] The inventors of this application have unexpectedly discovered that the relationship between the frequency (or percentage or number) of CD8 terminally differentiated effector T cells (CD8 TEMRA) in total CD8 T cells and the frequency (or percentage or number) of CD8 central memory T cells (CD8 TCM) in total CD8 T cells (e.g., expressed as the ratio of CD8 TEMRA to CD8 TCM (CD8 TEMRA: CD8 TCM) or the ratio of CD8 TCM to CD8 TEMRA (CD8 TCM: CD8 TEMRA)) in biological samples from subjects can be used as a highly accurate, readily available, non-invasive cell index biomarker for the diagnosis or prognosis of PD (and preferably early to intermediate PD, more preferably early PD).

[0045] More specifically, the inventors of this application employed an unbiased systemic immunology approach to analyze over 700 combined immunological signatures in the blood of early to mid-stage PD patients and matched healthy controls. As demonstrated in the experimental section (which exemplifies certain representative embodiments of the invention), the inventors of this application demonstrated that the CD8 TEMRA to CD8 TCM ratio was significantly increased in early to mid-stage PD patients compared to healthy controls. Interestingly, receiver operating characteristic (ROC) analysis based on the CD8 TEMRA to CD8 TCM ratio showed that early to mid-stage PD patients could be distinguished very well from healthy controls, with an excellent area under the curve (AUC) of 0.8503. Notably, an outstanding AUC of 0.9470 was obtained based on the CD8 TEMRA to CD8 TCM ratio in patients diagnosed only within 5 years. ROC analysis is the most mature and widely accepted method for evaluating the potential of biomarker candidates (Mandrekar JN, Receiver Operating Characteristic Curve in Diagnostic Test Assessment. Journal of Thoracic Oncology, 2010, Vol. 5, No. 9, pp. 1315-1316). Generally, an AUC of 0.5 indicates no discriminatory power (i.e., the ability to diagnose patients with and without the disease or condition based on the test), 0.7 to 0.8 is considered acceptable, 0.8 to 0.9 is considered excellent, and above 0.9 is considered outstanding.

[0046] In view of the foregoing, a first aspect provides a method (e.g., in vitro or ex vivo method) for diagnosing, prognosing, or monitoring Parkinson's disease (PD) in a subject, the method comprising determining the ratio of CD8 TEMRA to CD8 TCM cells based on values ​​of CD8 TEMRA and CD8 TCM determined in a biological sample of the subject. In a particular embodiment, the method comprises detecting or determining the amounts of CD8 TEMRA and CD8 TCM in a biological sample from the subject. In a particular embodiment, the method comprises determining the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample from the subject. In a particular embodiment, the method comprises determining the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample from the subject and diagnosing or prognosing PD in the subject based on the determined ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA).

[0047] In a particular implementation, the method is used for detecting PD.

[0048] In a particular implementation, the method is a means of monitoring disease progression in a subject diagnosed with PD.

[0049] In related aspects, this document provides the use of CD8 TEMRA and CD8 TCM in biological samples from a subject for the diagnosis, prognosis, or monitoring of PD in said subject. The use of the ratio of CD8 TEMRA to CD8 TCM or the ratio of CD8 TCM to CD8 TEMRA in biological samples from a subject for the diagnosis, prognosis, or monitoring of PD in said subject is also provided. In a particular embodiment, the method further includes the steps of treating the subject with a drug effective against PD progression, and optionally assessing their efficacy by monitoring the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in biological samples from the subject. In a particular embodiment, if the subject has been diagnosed with PD, the subject is treated with the drug only.

[0050] Non-limiting examples of drugs (i.e., therapeutic agents) that are effective against PD or PD progression include levodopa (e.g., carbidopa / levodopa (co-careldopa)), carbidopa, amantadine, dopamine agonists, anticholinergics (such as bentropine or trihexyphenidyl), monoamine oxidase-B (Mao-B) inhibitors (such as safenamide, selegiline, or rasagiline), catechol-O-methyltransferase (COMT) inhibitors (such as entacapone, opiapone, or tocapone), or apomorphine, or any combination thereof (such as levodopa and carbidopa).

[0051] As used in this article, the term “Parkinson’s disease” or “PD” refers to a complex, progressive neurodegenerative disorder that affects both the motor and non-motor systems. PD is caused by the loss of nerve cells in the substantia nigra. This leads to a reduction in dopamine in the brain, which plays a crucial role in regulating movement and coordination. The exact cause of nerve cell loss is unclear, but it may involve both genetic and environmental factors. Symptoms of PD typically develop slowly, and as the disease progresses, non-motor symptoms become more common. The main symptoms of PD include involuntary tremors (shaking) in specific parts of the body, bradykinesia, and muscle stiffness and inflexibility. While there is currently no cure for PD, treatments can be used to help reduce the main symptoms and maintain quality of life for as long as possible. Such treatments include supportive therapies (such as physical therapy and occupational therapy), medications (such as levodopa / carbidopa), and, in some cases, brain surgery.

[0052] The terms “diagnosing” or “diagnosis” are common and easily understood in medical and clinical practice. With further interpretation and without limitation, as used herein, “diagnosis” refers to the process or act of identifying, determining, or concluding a subject’s disease or condition based on symptoms and signs and / or the results of various diagnostic procedures (e.g., from the presence, absence, and / or quantity of one or more characteristic biomarkers of the diagnosed disease or condition). As used herein, “diagnosing” a subject’s disease or condition may specifically mean that the subject has such a disease or condition, and therefore the subject is diagnosed with such a disease or condition. As taught herein, “not diagnosing” a subject’s disease or condition may specifically mean that the subject does not have such a disease or condition, and therefore is diagnosed without such a disease or condition. Even if a subject exhibits one or more common symptoms or signs reminiscent of such a disease or condition, the subject may be diagnosed without such a disease or condition.

[0053] The terms “prognosing” or “prognosis” generally refer to the expectation of the progression of a disease or condition and the prospects for recovery (e.g., likelihood, duration, and / or extent). A good or favorable prognosis for PD as taught herein typically encompasses the expectation of satisfactory partial or complete recovery from the disease or condition, preferably within an acceptable timeframe. A poor or unfavorable prognosis for PD as taught herein typically encompasses the expectation of inadequate recovery and / or unsatisfactory, slow recovery, or essentially no recovery, or even further deterioration.

[0054] As used in this article, the terms "CD8 effector memory RA T cells" and "T cells" are relevant to this context. EMRA "CD8 terminally differentiated effector T cells" or "CD8 TEMRA", "terminally differentiated CD8 T cells", "terminally differentiated cytotoxic T cells", "terminally differentiated cytotoxic T cells" or "CD8 cells reexpressing CD45RA" refer to the most differentiated subtype of human CD8⁺ T cells. CD8 TEMRA can be characterized by the presence or absence of certain proteins or peptides on their cell membranes. For example, CD8 TEMRA can be characterized as CD8+ cells, which are CD45RA... + CD45RO - CCR7 - CD45RA + CCR7 - CD45RA + CCR7 - CD27 -CD45RA + CD27 - CD45RO - CCR7 - CD45RO - CCR7 - CD27 - Or CD45RO - CD27 - However, it is not limited to this.

[0055] As used in this article, the terms "CD8 central memory T cells" and "T cells" are relevant to the overall meaning of the text. CM "CD8 TCM" or "long-lived central memory CD8 T cells" refers to T cells that possess both antigen-independent homeostatic proliferation and vigorous proliferation capacity after antigen re-exposure. CD8 TCMs can be characterized by the presence or absence of certain proteins or peptides on their cell membranes. For example, CD8 TCMs can be characterized by CD45RO + CCR7 + CD27 + CD45RO + CD62L + CD27 + CD45RO + CD62L + CCR7 + CD27 + CD45RO + CCR7 + CD45RO + CD27 + CD45RO + CD62L + CD45RA - CD62L + CCR7 + CD27 + CD45RA - CD62L + CD27 + CD45RA - CCR7 + CD27 + CD45RA - CCR7 +、 CD45RA - CD27 + Or CD45RA - CD62L + However, it is not limited to this.

[0056] The term "sample" or "biological sample" as used throughout this specification includes any biological sample obtained (isolated, removed) from a subject. Samples may include, but are not limited to, whole blood, plasma, serum, whole blood cells, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), saliva, urine, feces, tears, sweat, sebum, organ tissue, exhaled fluid, nipple aspiration, catheter irrigation fluid, tumor exudate, interstitial fluid, synovial fluid, cerebrospinal fluid, lymph, fine-needle aspiration, amniotic fluid, any other body fluid, exudate or secretion, cell lysate, cellular secretory products, inflammatory fluid, semen, and vaginal secretions. Preferably, the sample can be readily obtained by non-invasive or minimally invasive methods (e.g., blood collection ('liquid biopsy'), urine collection, fecal collection, tissue (e.g., tumor tissue) biopsy, or fine-needle aspiration), thereby allowing the sample to be provided / removed / isolated from the subject. As used herein, the term "tissue" encompasses all types of body cells, including organ cells, but also includes blood and the other body fluids described above. The tissue may be derived from a living subject or may be cadaveric tissue. In a particular embodiment, the sample is a dried blood spot (DBS) or any other trace sample of capillary blood.

[0057] In a specific implementation, the biological sample is a body fluid sample, preferably a peripheral whole blood sample.

[0058] Preferably, the biological sample is a fresh sample or a frozen sample, with fresh samples being preferred. In a particular embodiment, the biological sample is obtained from the subject up to 24 hours, up to 20 hours, up to 16 hours, up to 12 hours, up to 8 hours, up to 4 hours, or up to 1 hour before detecting or measuring CD8 TEMRA and CD8 TCM and / or their ratio.

[0059] Particularly useful samples are those that are known to contain, or expected to contain, or predicted to contain, or are known to potentially contain, or are expected to potentially contain, peripheral blood mononuclear cells (PBMCs).

[0060] In a particular implementation, the biological sample (and / or reference sample) is a whole blood sample.

[0061] In certain implementations, the biological sample (and / or reference sample) comprises, is substantially composed of, or is composed of peripheral blood mononuclear cells (PBMCs). The sample may be obtained from the subject in any manner commonly used in a clinical setting to obtain a sample containing the desired cells, peptides, proteins, polypeptides, or nucleic acids (including RNA). If desired, the sample may be mixed with liquids or purified or amplified or otherwise processed. For example, the sample may be processed in one or more purification steps to increase the purity of the desired cells, peptides, proteins, polypeptides, or nucleic acids in the sample, or they may be examined without any purification steps. Any peptide, protein, polypeptide, or nucleic acid sample obtained from such samples, in purified or unpurified form, may be utilized in the methods taught herein. CD8 TEMRA and CD8 TCM, or their ratio, may be measured first from whole blood samples or from PBMCs, or without cell separation. As used herein, the term "purified" in relation to markers, cells, peptides, polypeptides, proteins, or nucleic acids does not require absolute purity. Conversely, it indicates that such markers, cells, peptides, polypeptides, proteins, or nucleic acids are in discrete environments where their abundance relative to other cells or analytes is greater than their abundance in biological samples.

[0062] In a particular implementation, PBMCs are isolated from whole blood samples, for example by gradient centrifugation, before the ratio of CD8 TEMRA to CD8 TCM is determined.

[0063] The terms “subject,” “individual,” or “patient” are used interchangeably throughout the specification and generally and preferably refer to humans, but may also encompass references to non-human animals, preferably warm-blooded animals, and even more preferably mammals, such as non-human primates, rodents, canines, felines, equines, sheep, pigs, etc. The term “non-human animal” includes all vertebrates, such as mammals (e.g., non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle), and non-mammals (e.g., chickens, amphibians, reptiles, etc.). In some embodiments, the subject is a non-human mammal. In some preferred embodiments of the methods or uses taught herein, the subject is a human subject. In other embodiments, the subject is an experimental animal or animal substitute used as a disease model. The term does not indicate a specific age or sex. Thus, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be included. Examples of subjects include humans, dogs, cats, cattle, goats, and mice. The term "subject" is also intended to include genetically modified species. In a particular implementation, the subject is a human subject.

[0064] Suitable subjects may include, but are not limited to, subjects who present to a physician with one or more symptoms of PD (e.g., early to mid-stage PD) (e.g., motor symptoms), or subjects who have been diagnosed with PD (optionally receiving PD treatment).

[0065] Based on the Hoehn and Yahr scale (Hoehn and Yahr, Parkinsonism: Onset, Progression and Mortality, 1967, 17(5): 427-42), PD can be divided into five stages in terms of clinical disability. In this scale, stages 1 and 2 represent early PD, stages 2 and 3 represent intermediate PD, and stages 4 and 5 represent late PD. Those skilled in the art will understand that other widely accepted classification methods can be used to define the stages of PD.

[0066] In certain embodiments, the subjects are those with early to intermediate stage PD (preferably early PD). In certain methods, the subjects are those with stage 1 or 2 PD (preferably stage 1 PD) according to the Hoehn and Yahr scale. Therefore, in certain embodiments, the methods taught herein are methods for diagnosing, prognosing, or monitoring early to intermediate stage PD (preferably early PD).

[0067] In a particular implementation, the subject is at most 75 years old or at most 70 years old. In a particular implementation, the subject is between 50 and 75 years old, between 50 and 70 years old, between 55 and 70 years old, and preferably between 60 and 70 years old.

[0068] The methods taught herein allow for highly accurate diagnosis or prognosis of PD. Furthermore, the inventors of this application have demonstrated AUC values ​​as high as 0.93 in female subjects. Therefore, in a particular embodiment, the subject is female.

[0069] In a specific implementation, the subject is serologically positive for cytomegalovirus (CMV).

[0070] In a particular implementation, such as if the method taught herein is a method for prognosticating or monitoring PD in a subject, the biological samples are obtained from the subject at two or more different time points, and the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) is determined at different time points.

[0071] In specific implementations, methods as taught herein include the steps of detecting CD8 TEMRA and CD8 TCM in a subject's biological sample, and optionally (e.g., before determining the ratio) measuring the quantity (i.e., amount) of CD8 TEMRA and CD8 TCM in the subject's biological sample. Any existing, available, or conventional isolation, detection, and quantification methods may be used herein to measure the presence or absence (e.g., readout of presence or absence; or detectable or undetectable amounts) and / or quantity (e.g., readout as absolute or relative quantity, such as absolute or relative concentration). For example, such methods may include flow cytometry for cells.

[0072] Methods for detecting CD8 TEMRA and CD8 TCM are known in the art, and said methods include, but are not limited to, cytometry, such as conventional flow cytometry or spectral flow cytometry or imaging flow cytometry, mass spectrometry flow cytometry (also known as CyTOF), or other antibody-based single-cell analysis methods or other available methods that combine antibody detection within the same single cell with the measurement of both other types of molecules (e.g., RNA). For example, in a particular implementation, the method taught herein includes the step of detecting CD8+ T cells, said CD8+ T cells being CD45RA. + CD45RO - CCR7 - CD45RA + CCR7 - CD45RA + CCR7 - CD27 - CD45RA + CD27 - CD45RO - CCR7- CD45RO - CCR7 - CD27 - Or CD45RO - CD27 - For example, in a particular implementation, the method taught herein includes the step of detecting CD8+ T cells, said CD8+ T cells being CD45RO. + CCR7 + CD27 + CD45RO + CD62L + CD27 + CD45RO + CD62L + CCR7 + CD27 + CD45RO + CCR7 + CD45RO + CD27 + CD45RO + CD62L + CD45RA - CD62L + CCR7 + CD27 + CD45RA - CD62L + CD27 + CD45RA - CCR7 + CD27 + CD45RA - CCR7 + CD45RA - CD27 + Or CD45RA - CD62L + In a particular embodiment, the method includes using reagents specifically for detecting CD8 TCM cells and CD8 TEMRA cells based on the biomarkers listed above. In a particular embodiment, the method includes using reagents specifically for detecting CD45RO, CD45RA, CCR7, and CD27.

[0073] In certain implementations, the amount or number of CD8 TEMRA and CD8 TCM can be determined using flow cytometry (FCM) (e.g., multi-panel multicolor FCM).

[0074] The terms “quantity,” “amount,” “number,” and “level” are synonymous and are generally well understood in the art. Terms used herein may specifically refer to absolute quantification of cells, molecules, or analytes in a sample, or relative quantification of cells, molecules, or analytes in a sample, i.e., relative to another value (e.g., relative to a reference value as taught herein), or a range of values ​​representing baseline expression levels of a biomarker. These values ​​or ranges can be obtained from a single patient or a group of patients.

[0075] In a particular implementation, the method further includes determining the ratio of CD8 TEMRA to regulatory T cells (CD8 Treg) (or the ratio of CD8 Treg to CD8 TEMRA).

[0076] In a particular implementation, the method does not include measuring any biomarkers other than the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA).

[0077] The inventors of this application have discovered that the ratio of CD8 TEMRA to CD8 TCM in biological samples from subjects with PD is significantly different (or vice versa) compared to biological samples from healthy subjects (or subjects without PD).

[0078] Therefore, in certain embodiments, the methods taught herein may include the step of comparing the determined ratio of CD8 TEMRA to CD8 TCM (or vice versa) with a reference (e.g., a reference sample, a threshold, or a cutoff value). Similarly, in certain embodiments, the method includes comparing the determined ratio of CD8 TEMRA to CD8 TCM (or vice versa) with a reference, and further determining whether the ratio changes (or differs from) the reference when compared. In certain embodiments, the reference sample is a sample from a healthy subject or a group of healthy subjects, or a sample from a subject (or group of subjects) known not to have PD. In certain embodiments, if the methods taught herein are used to predict the survival or prognosis of a subject's PD, the reference may be a sample taken from the subject (i.e., the same subject) at an earlier time point. In certain embodiments, if the methods taught herein are used to predict the survival or prognosis of a subject's PD, the reference may be a subject or group of subjects known to have a favorable or unfavorable PD prognosis.

[0079] Thresholds or cutoff values ​​commonly known in the art can be selected to provide the chosen accuracy, sensitivity, and / or specificity of the prediction method, such as at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95% accuracy, sensitivity, and / or specificity.

[0080] For example, receiver operating characteristic (ROC) curve analysis can be used to select the optimal threshold or cutoff for the number of a given number of biomarkers for clinical use in this diagnostic test, based on acceptable overall accuracy, sensitivity and / or specificity, or relevance metrics that are well-known themselves (e.g., positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index (i.e., sensitivity + specificity - 1), or similar metrics).

[0081] For example, an optimal threshold or cutoff value can be selected for each individual biomarker as a local extremum of the receiver operating characteristic (ROC) curve, i.e., the point at which the distance to the local maximum diagonal is, as described in Robin X., Panelomi X: atreshold-based algorithm to create panels of biomarkers, 2013, Translational Proteomics, 1(1): 57-64.

[0082] Those skilled in the art will understand that providing a precise threshold or cutoff value is not important. A relevant threshold or cutoff value can be obtained by correlating sensitivity and specificity with the sensitivity / specificity of any threshold or cutoff value.

[0083] The diagnostic engineer or clinician determines which levels of positive predictive value / negative predictive value / sensitivity / specificity are desirable, and how much of a decrease in positive or negative predictive value is acceptable. The chosen threshold or cutoff level may depend on other diagnostic parameters used by the diagnostic engineer in conjunction with this method.

[0084] In a particular implementation, the threshold or cutoff value can vary depending on the subgroup of PD patients, such as male / female or early PD patients (e.g., diagnosed within 5 years of disease onset, such as within 5 years of the onset of the first motor symptom).

[0085] The precise threshold or cutoff value for the optimal ratio between CD8 TEMRA and CD8 TCM, for various subgroups of subjects being tested, can be chosen based on the specific diagnostic or prognostic needs of the responsible neurologist or other clinician. Therefore, several established methods can be chosen to define the optimal cutoff value by maximizing the trade-off between sensitivity and specificity, such as the Youden index (as described elsewhere in this document). In some implementations, such as when the methods taught herein are used to diagnose PD, an acceptable specificity (e.g., >= 85%) can be fixed first, and then sensitivity can be maximized. In other implementations, such as when the methods taught herein are used for prognosticating PD, an acceptable sensitivity (e.g., >= 85%) can be fixed first, and then specificity can be maximized. In some implementations, the appropriate cutoff value for decision-making can be determined simply by setting the expected likelihood ratio (e.g., >= 3) or odds ratio.

[0086] For example, for CD8 TEMRA: the threshold or cutoff value for the CD8 TCM ratio can be one of those defined in any of Tables 4-7.

[0087] For example, based on the maximum Oden index (e.g., sensitivity of 88.89%, specificity of 75%, and likelihood ratio of 3.556), the threshold or cutoff value for the CD8 TEMRA:CD8 TCM ratio could be approximately 14.3. For example, if the subject was clinically diagnosed within 5 years, the cutoff value for the CD8 TEMRA:CD8 TCM ratio could be approximately 19.2 based on the maximum Oden index (e.g., sensitivity of 100%, specificity of 83.33%, and likelihood ratio of 6). For example, if the subject is female, the cutoff value for the CD8 TEMRA:CD8 TCM ratio could be approximately 13.2 based on the maximum Oden index (e.g., sensitivity of 100%, specificity of 80%, and likelihood ratio of 5). For example, if the subject is male, the cutoff value for the CD8 TEMRA:CD8 TCM ratio could be approximately 19.2 based on the maximum Oden index (e.g., sensitivity of 78.95%, specificity of 78.57%, and likelihood ratio of 3.684).

[0088] In a particular implementation, the method does not include comparing any biomarkers other than the ratio of CD8TEMRA to CD8 TCM (or vice versa) in the biological sample from the subject to a reference.

[0089] As explained, the methods, uses, or products of this application may involve finding a difference or no difference between the ratio of CD8 TEMRA to CD8 TCM in a biological sample from a subject (or vice versa) and the ratio of CD8 TEMRA to CD8 TCM in a reference (e.g., a reference sample) (or vice versa), or finding a difference or no difference between the ratio of CD8 TEMRA to CD8 TCM in a biological sample from a subject and a threshold or cutoff value.

[0090] The “difference” between the first and second values ​​can generally encompass any direction (e.g., increase: first value > second value; or decrease: first value < second value) and any degree of change. In this case, the first value could be the ratio of CD8 TEMRA to CD8 TCM in a biological sample from a subject suspected of having PD (or vice versa), and the second value could be a reference (e.g., a reference value). Alternatively, the first value could be the ratio of CD8 TEMRA to CD8 TCM in a biological sample from a subject suspected of having PD (or vice versa), and the second value could be a threshold or cutoff value.

[0091] For example, the difference may cover (but is not limited to) a reduction of at least about 10% (about 0.9 times or less), or at least about 20% (about 0.8 times or less), or at least about 30% (about 0.7 times or less), or at least about 40% (about 0.6 times or less), or at least about 50% (about 0.5 times or less), or at least about 60% (about 0.4 times or less), or at least about 70% (about 0.3 times or less), or at least about 80% (about 0.2 times or less), or at least about 90% (about 0.1 times or less) relative to the second value used for comparison.

[0092] For example, the difference may cover (but is not limited to) an increase of at least about 10% (about 1.1 times or more), or at least about 20% (about 1.2 times or more), or at least about 30% (about 1.3 times or more), or at least about 40% (about 1.4 times or more), or at least about 50% (about 1.5 times or more), or at least about 60% (about 1.6 times or more), or at least about 70% (about 1.7 times or more), or at least about 80% (about 1.8 times or more), or at least about 90% (about 1.9 times or more), or at least about 100% (about 2 times or more), or at least about 150% (about 2.5 times or more), or at least about 200% (about 3 times or more), or at least about 500% (about 6 times or more), or at least about 700% (about 8 times or more), etc., relative to the second value used for comparison.

[0093] Preferably, a difference can refer to a statistically significant observed change. For example, a difference can refer to an observed change that falls outside the error limits of a reference value in a given population (e.g., expressed as a standard deviation or standard error, or as a predetermined multiple thereof, such as ±1×SD, ±2×SD, or ±3×SD, or ±1×SE, ±2×SE, or ±3×SE). A difference can also refer to a value that falls outside a reference range defined by values ​​in a given population (e.g., outside a range that includes ≥40%, ≥50%, ≥60%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, or even ≥100% of the values ​​in said population).

[0094] In another implementation, if the observed change exceeds a given threshold or cutoff value, a difference can be inferred.

[0095] The methods, uses, or products of this application may also relate to attributing any finding of difference or lack thereof between the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample from a subject and a given reference value or threshold to a diagnosis or prognosis of PD. The reference value may be the ratio of CD8 TEMRA to CD8 TCM in the biological sample determined in a reference sample, or a threshold or cutoff value.

[0096] In a particular implementation, observing a difference between the CD8 TEMRA to CD8 TCM ratio (or the CD8 TCM to CD8 TEMRA ratio) and a reference leads to the conclusion that the subject's diagnosis or prognosis of PD differs from that represented by the reference. Similarly, in a particular implementation, when there is no difference between the CD8 TEMRA to CD8 TCM ratio (or the CD8 TCM to CD8 TEMRA ratio) and a reference, the absence of such a difference leads to the conclusion that the subject's diagnosis or prognosis of PD is substantially the same as or identical to that represented by the reference.

[0097] Different references may represent the diagnosis or prognosis of PD in the subject, or they may not exist.

[0098] In certain implementations, the references used in the methods taught herein are determined from biological samples from healthy subjects or a group of healthy subjects (e.g., subjects or a group of subjects known not to have PD).

[0099] In certain implementations, the references used in the methods taught herein are determined from biological samples from subjects or a group of subjects known to have PD.

[0100] In certain embodiments, the references used in the methods taught herein are determined from biological samples from subjects or groups known to have a favorable PD outcome. In certain embodiments, the references used in the methods taught herein are determined from biological samples from subjects or groups known to have an unfavorable PD outcome.

[0101] In a particular implementation, the method includes the following steps:

[0102] (a) Determine the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in biological samples from subjects;

[0103] (b) Compare the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) determined in (a) with a reference (e.g., a reference representing a known PD diagnosis or prognosis);

[0104] (c) Diagnose, predict or monitor the PD of the subject based on the comparison in step (b).

[0105] It should be understood that the ratio of CD8 TEMRA to CD8 TCM is mentioned.

[0106] In a particular implementation, the method includes the following steps:

[0107] (i) Determine the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in biological samples from subjects;

[0108] (ii) Compare the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) determined in (i) with a reference (e.g., a reference representing a known PD diagnosis or prognosis);

[0109] (iii) The ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) measured in (i) is found to be different from or not different from the reference; and

[0110] (iv) Attributing the findings, whether different or not, to a specific diagnosis or prognosis of the subject's PD.

[0111] In certain implementations, the ratio of CD8TEMRA to CD8 TCM may be increased compared to a reference as described elsewhere herein (i.e., relative to a reference). This increased ratio may allow for the diagnosis or prognosis of PD in the subject.

[0112] In a particular embodiment, the method includes determining whether the CD8 TEMRA to CD8 TCM ratio is increased when compared to a reference (e.g., a sample from a healthy subject). In a particular embodiment, for example, if the reference corresponds to the CD8 TEMRA to CD8 TCM ratio in a biological sample (e.g., a healthy sample) unaffected by PD, then a significant increase in the CD8 TEMRA to CD8 TCM ratio determined in (i) compared to the reference allows for the diagnosis or prognosis of PD in the subject. In a particular embodiment, for example, if the reference corresponds to the CD8 TEMRA to CD8 TCM ratio in a biological sample (e.g., a healthy sample) unaffected by PD, then no significant difference in the CD8 TEMRA to CD8 TCM ratio determined in (i) compared to the reference allows for the conclusion that the subject does not have PD.

[0113] In a particular implementation, for example if the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample affected by PD, then the lack of significant difference between the CD8 TEMRA to CD8 TCM ratio determined in (i) and the reference allows for the diagnosis or prognosis of PD in the subject.

[0114] In a particular embodiment, the method includes determining whether the ratio of CD8 TCM to CD8 TEMRA is reduced when compared to a reference (e.g., a sample from a healthy subject). In a particular embodiment, if the reference corresponds to the ratio of CD8 TCM to CD8 TEMRA in a biological sample unaffected by PD (e.g., a healthy sample), then a significant reduction in the ratio of CD8 TEMRA to CD8 TCM determined in (i) compared to the reference allows for the diagnosis or prognosis of PD in the subject; or in a particular embodiment, if the reference corresponds to the ratio of CD8 TCM to CD8 TEMRA in a biological sample affected by PD, then no significant difference in the ratio of CD8 TCM to CD8 TEMRA determined in (i) compared to the reference allows for the diagnosis or prognosis of PD in the subject.

[0115] In a particular implementation, if the method taught herein is used for the prognosis of a subject's PD, the reference may be a sample taken from the subject at an earlier time point. In a particular implementation, if the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a sample taken from the subject at an earlier time point, then a significant increase in the CD8 TEMRA to CD8 TCM ratio determined in (i) compared to the reference allows for an unfavorable PD prognosis for the subject. In a particular implementation, if the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a sample taken from the subject at an earlier time point, then a significant decrease in the CD8 TEMRA to CD8 TCM ratio determined in (i) compared to the reference allows for a favorable PD prognosis for the subject.

[0116] In another implementation, if the observed change exceeds a given threshold or cutoff value, a difference can be inferred.

[0117] For example, a diagnosis of PD can be made if the ratio of CD8 TEMRA to CD8 TCM measured in (i) is greater than approximately 14.3. For example, if the subject undergoes a clinical diagnosis within 5 years (e.g., within 5 years of disease onset, such as within 5 years of the first sign of motor symptoms), a diagnosis of PD can be made if the ratio of CD8 TEMRA to CD8 TCM measured in (i) is greater than approximately 19.2. For example, if the subject is female, a diagnosis of PD can be made if the ratio of CD8 TEMRA to CD8 TCM measured in (i) is greater than approximately 13.2. For example, if the subject is male, a diagnosis of PD can be made if the ratio of CD8 TEMRA to CD8 TCM measured in (i) is greater than approximately 19.2.

[0118] On the other hand, kits are provided (particularly kits for the diagnosis, prognosis, or monitoring of PD in subjects), said kits comprising: (a) a tool specifically adapted to determine the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample from a subject, and (b) optionally a reference for said ratio or a tool for establishing said reference, for example, wherein said reference represents a known diagnosis or prognosis of PD. Preferably, the tool (a) and optionally the reference (b) are the only biological reagents present in said kit. In a particular embodiment, said kit comprises: (a) a tool specifically adapted to determine the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample from a subject, and (b) a reference for said ratio, which allows for the diagnosis, prognosis, or monitoring of PD based on said ratio determined from the subject's sample. In a particular embodiment, said kit comprises a tool specifically adapted to determine the values ​​of CD8 TEMRA and CD8 TCM in a biological sample from a subject. Preferably, the kit contains only the specifically applicable reagent and a reference for the ratio.

[0119] As used throughout this specification, the terms "kit of components" and "kit" refer to a product containing components required to perform the specified method, which are packaged to allow for their transport and storage. Suitable materials for packaging the components contained in a kit include crystals, plastics (e.g., polyethylene, polypropylene, polycarbonate), bottles, flasks, vials, ampoules, paper, envelopes, or other types of containers, carriers, or supports. When a kit contains multiple components, at least a subset of the components (e.g., two or more of a plurality of components) or all of the components may be physically separated, for example, contained in or on separate containers, carriers, or supports. The components contained in the kit may be sufficient or insufficient to perform the specified method, such that external reagents or substances may or may not be necessary for performing the method separately. Typically, kits are used in conjunction with standard laboratory equipment, such as liquid handling equipment, environmental (e.g., temperature) control equipment, analytical instruments, etc. In addition to the isolated oligonucleotide sets as taught herein (optionally provided on an array or microarray), the kits of this application may also include some or all of the following: solvents, buffers (e.g., but not limited to histidine buffer, citrate buffer, succinate buffer, acetate buffer, phosphate buffer, formate buffer, benzoate buffer, TRIS (tris(hydroxymethyl)-aminomethane) buffer or maleate buffer, or mixtures thereof), enzymes (e.g., but not limited to thermostable DNA polymerases), detectable tags, assay reagents, and control preparations (positive and / or negative) useful in the specified methods. Typically, the kits may also include instructions for use, such as those on a printed insert or a computer-readable medium. When used in the context of this application, these terms may be used interchangeably with the term "manufactured article," which broadly covers any artificially manufactured tangible structure product.

[0120] In a particular embodiment, the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample unaffected by PD (e.g., a healthy sample), or the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample affected by PD.

[0121] In a particular implementation, the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in biological samples from subjects with known favorable or unfavorable prognoses.

[0122] In a particular implementation, the kit contains (or the reference corresponds to) a list of possible thresholds or cutoff values ​​having corresponding sensitivity, specificity, and likelihood ratios for PD subjects or their different subgroups (e.g., male-only, female-only, or early PD subjects (e.g., within 5 years, such as within 5 years after disease onset, or within 5 years after the first symptom of motor symptoms). The list may be a table provided in any of Tables 4-7 in the Examples section.

[0123] In a particular implementation, the kit includes one or more thresholds or cutoff values ​​as described elsewhere herein.

[0124] In certain embodiments, the tools specifically designed for determining the CD8 TEMRA to CD8 TCM ratio include one or more binders that allow CD8 TEMRA and CD8 TCM to be distinguished from each other, and optionally allow CD8 TEMRA and CD8 TCM to be distinguished from other CD8+ T cells. Such binders are known in the art and include, but are not limited to, antibodies.

[0125] For example, the tools include:

[0126] - A binding agent that specifically binds to CD3;

[0127] - A binding agent that specifically binds to CD4;

[0128] - A binder that specifically binds to CD8;

[0129] - A binder that specifically binds to CD45RO;

[0130] - A binder that specifically binds to CD45RA;

[0131] - A binder that specifically binds to CD27;

[0132] - A binder that specifically binds to CD62L; and / or

[0133] - A binder that specifically binds to CCR7;

[0134] Preferably

[0135] - A binder that specifically binds to CD8;

[0136] - A binder that specifically binds to CD45RO;

[0137] - A binder that specifically binds to CD45RA;

[0138] - A binder that specifically binds to CD27;

[0139] - A binder that specifically binds to CD62L; and / or

[0140] - A binder that specifically binds to CCR7.

[0141] Preferably, the kit comprises or consists of: a binding agent specifically binding to CD45RO, a binding agent specifically binding to CD45RA, a binding agent specifically binding to CCR7, and a binding agent specifically binding to CD27. In a particular embodiment, the kit comprises or consists of: a binding agent specifically binding to CCR7 and a binding agent specifically binding to CD27, and optionally other reagents for selecting CD8 T cells. In another embodiment, the kit comprises or consists of: a binding agent specifically binding to CD45RO and a binding agent specifically binding to CCR7, or a binding agent specifically binding to CD45RA⁺, and a binding agent specifically binding to CCR7. In yet another embodiment, the kit comprises a binding agent specifically binding to CD45RO and a binding agent specifically binding to CD27, or a binding agent specifically binding to CD45RA, and a binding agent specifically binding to CD27.

[0142] In a particular implementation, the kit does not contain any tools specifically designed to determine biomarkers other than the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA).

[0143] On the other hand, the kits, as taught herein, are provided for use in the diagnosis, prognosis, or monitoring of PD based on the determination of the ratio of CD8 TEMRA to CD8 TCM in a subject's biological sample.

[0144] On the other hand, it provides the use of the kits taught herein in methods taught herein (e.g., ex vivo methods taught herein) for the diagnosis, prognosis, or monitoring of PD in subjects.

[0145] On the other hand, this article provides methods for diagnosing, prognosing, and / or monitoring PD in subjects, as well as for treatment, including:

[0146] (a) Determination of CD8 TEMRA and CD8 TCM in biological samples from the subjects;

[0147] (b) Diagnostic or prognostic assessment of PD in the subjects based on CD8 TEMRA and CD8 TCM detection; and

[0148] (c) Administering a therapy for PD to the subject who has been diagnosed or is presumed to have PD, such as administering an effective amount of a PD treatment agent to the subject.

[0149] In a particular embodiment, detecting CD8 TEMRA and CD8 TCM includes determining (e.g., measuring) the amounts of CD8 TEMRA and CD8 TCM. In a particular embodiment, the method includes determining the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TEMRA to CD8 TCM) in a biological sample from a subject.

[0150] On the other hand, a PD therapeutic agent is provided for use in a method for treating PD in a subject, wherein the method includes diagnosing, prognosing, or monitoring the subject's PD using methods as taught herein, and administering the PD therapeutic agent to the subject if the subject is diagnosed with PD.

[0151] The terms "treat" or "treatment" encompass therapeutic treatment of an already developed disease or condition, such as therapy for developed Parkinson's disease (PD), as well as preventative or preventive measures aimed at preventing or reducing the chance of undesirable suffering, such as preventing the onset, development, and progression of PD. Beneficial or desired clinical outcomes may include, but are not limited to, relief of one or more symptoms or one or more biomarkers, reduction of disease severity, stabilization (i.e., non-exacerbation) of the disease state, delay or slowing of disease progression, improvement or mitigation of the disease state, etc.

[0152] As used herein, the term "therapeuticly effective dose" refers to the amount of an active compound or agent that a researcher, veterinarian, physician, or other clinician is seeking to elicit a biological or pharmaceutical response in a subject, which may include, in particular, the relief of symptoms of a disease or condition being treated. Methods known in the art for determining therapeutic and preventatively effective doses for immunotherapeutic agents or pharmaceutical formulations as taught herein are available.

[0153] The products and methods taught herein allow for the administration of a therapeutically effective amount of a PD therapeutic agent to a subject suffering from PD who would benefit from such treatment. As used herein, the term "therapeuticly effective amount" refers to the amount of an active compound or agent that a surgeon, researcher, veterinarian, physician, or other clinician is seeking to elicit a biological or pharmacological response in a subject, which may include, in particular, relief of symptoms of the disease or condition being treated. Methods for determining the therapeutically effective dose of a PD therapeutic agent are known in the art.

[0154] As used herein, the term “therapeutic effective dose” refers to the amount of PD treatment agent that, when administered, produces a positive therapeutic response relative to the treatment of a patient with PD.

[0155] The appropriate therapeutic dose and dosing regimen for PD treatment agents can be determined by a qualified physician based on the nature of PD treatment, disease status and severity, as well as the patient's age, body size and condition.

[0156] Computer program

[0157] On the other hand, a computer program product for use in conjunction with a computer having a processor and memory connected to the processor is provided. The computer program product includes a computer-readable storage medium thereon on which a computer program mechanism is encoded, wherein the computer program mechanism can be loaded into the memory of the computer and cause the computer to perform the methods taught herein.

[0158] In another related aspect, the present invention relates to a system comprising:

[0159] (a) A computer data repository comprising references (e.g., reference values ​​or thresholds) for the ratio of said CD8 TEMRA to CD8 TCM, such as reference values ​​representing known diagnoses or prognoses of PD; and

[0160] (b) A computer system programmed to access the data repository and, in samples from subjects with PD symptoms or diagnosed with PD, to diagnose or predict PD in the subjects using information from the data repository combined with information about the ratio of CD8 TEMRA to CD8 TCM.

[0161] On the other hand, a computer-executed method is provided for diagnosing, prognosing, or monitoring PD in a subject, the method comprising determining, by a processor, the ratio of CD8 TEMRA to CD8 TCM (or the ratio of CD8 TCM to CD8 TEMRA) in a biological sample from the subject.

[0162] In a particular implementation, the method includes providing quantitative values ​​of CD8 terminally differentiated effector T cells (CD8 TEMRA) and CD8 central memory T cells (CD8 TCM) in a biological sample from a subject.

[0163] In a particular embodiment, the computer-executed method may include the steps of: determining the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM), as determined in the biological sample; and generating a diagnostic or prognostic output by the processor based on the ratio obtained in the determining step.

[0164] In certain embodiments, the computer-executed method may further include a step of comparing the CD8 TEMRA to CD8 TCM ratio obtained in the determining step with a reference. In certain embodiments, the computer-executed method may further include a step of finding whether the determined CD8 TEMRA to CD8 TCM ratio differs from or does not differ from the reference value. In certain embodiments, the computer-executed method may further include a step of generating a diagnostic or prognostic output based on the finding of difference or no difference, wherein the diagnostic output includes a prediction of the likelihood of a condition present in the subject. In certain embodiments, the computer-executed method may further include a step of displaying the diagnostic output on a user interface for viewing by a healthcare professional or physician. In certain embodiments, the computer-executed method may further include a step of updating the machine learning model using feedback from a healthcare professional or physician regarding the accuracy of the diagnostic or prognostic output, thereby improving future diagnostic predictions. These different steps of comparison, finding difference or no difference, and generating the above-described output are typically performed by a processor.

[0165] Another related aspect of the invention relates to a method for diagnosing, prognosing, or monitoring PD in a subject who has symptoms of PD or has been diagnosed with PD, the method comprising the following steps:

[0166] (a) Receive data from samples from subjects representing the ratio of CD8 TEMRA to CD8 TCM;

[0167] (b) Access a data repository on a computer that includes references (e.g., reference values ​​or thresholds) for the ratio of CD8 TEMRA to CD8 TCM, such as reference values ​​representing known diagnoses or prognoses of PD; and

[0168] (c) The data received in (a) is compared with a reference in the data repository on the computer, and the subject’s PD is diagnosed or prognosed based on the comparison.

[0169] In a particular implementation, taking into account the predictions, determining what actions to take (particularly by clinicians) is performed by the computer. More preferably, the computer, preferably substantially in real time, reports the actions to be taken (i.e., generates an electronic report of the actions to be taken).

[0170] While the invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be apparent from the foregoing description. Therefore, all such alternatives, modifications, and variations are intended to be covered within the spirit and scope of the appended claims.

[0171] The aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples. Example

[0172] Example 1. The CD8 TEMRA to CD8 TCM ratio allows for highly accurate diagnosis or prognosis of early to mid-stage PD.

[0173] The inventors of this application applied a systems immunology approach to comprehensively analyze the peripheral immune systems of early to mid-stage PD patients and matched healthy controls (HC). By focusing on early to mid-stage patients, the inventors inferred that they could have a higher probability of identifying peripheral immune cells that initiate the pathogenesis of PD (especially idiopathic PD (iPD)) rather than those peripheral immune cells that make secondary responses to the manifestation of PD pathological events.

[0174] In the discovery study, the inventors of this application systematically analyzed various immune subtypes and their functional status in 28 PD patients (25 iPD patients aged 60-70 years and 3 hereditary PD patients with GBA or PINK1 mutations) and 24 matched healthy controls (HC) ('PD patients' are abbreviated as PD below). This was achieved by studying 37 different innate and adaptive immune subtypes and more than 700 combined T cell characteristics, using a 35-marker spectrometry flow cytometry (also known as time-of-flight flow cytometry or CyTOF) panel and five multicolor flow cytometry (FCM) panels consisting of 33 lineage and functional T cell markers. Figure 1 A. Tables 1 and 2). The inventors of this application recruited participants from the ongoing nationwide Luxembourg Parkinson's study (https: / / parkinson.lu / research-participation / luxembourg-parkinsons-study), which has over 800 PDs and 800 HCs, and controlled for several major confounding factors, drugs, and comorbidities known to affect the immune system to ensure that their observations were PD-specific (see details). Figure 1(A and the following section, “Cohort Design”). Furthermore, the inventors of this application narrowed the patients to those with early to mid-stage disease [Hoehn and Yahr (H&Y) staging scale: mean = 2.3, range 1.5 to 3.0; most of them ≤2.5, except for five participants at grade 3]. Most of the selected patients had a disease duration of 10 years from clinical diagnosis, with three patients having disease durations of 12, 13, and 19 years, respectively. The inventors of this application focused on the relatively narrow age window (60–70 years) in the PD and corresponding HC groups. To make the immunoassays comparable at such advanced ages, the inventors of this application only invited HC and PD subjects as participants for fresh blood sampling if they were seropositive against CMV (cytomegalovirus) IgG. The inventors of this application only invited CMV seropositive participants for technical and epidemiological reasons. Technically, the inventors of this application could not rule out the possibility that some CMV-negative participants might become CMV seropositive during the time interval between the previous serological sampling and the current fresh blood sampling. Secondly, based on large-scale studies of CMV seropositivity in adjacent regions, most older adults were found to be CMV seropositive. Selecting CMV seronegative individuals within this age range would further severely limit the availability of suitable samples. Therefore, the inventors of this application decided to first analyze a more representative group of participants in their sixties.

[0175] Early to mid-stage iPD exhibits an increased effector spectrum in CD8 T cells.

[0176] Since the inventors of this application had already demonstrated substantial changes in T cell compartments in whole blood of iPD patients using CyTOF analysis, they subsequently used five FCM panels with a total of 33 T cell-related biomarkers (combinations of which produce ~700 signatures) for further T cell analysis. In this way, the inventors of this application were able to assess not only phenotypic biomarkers and the proportions of different T cell subsets, but also the functional status of different subtypes (Table 2). Based on ~700 different combinations of PCA, a unique immunofingerprint was determined in iPD compared to HC (except for one PD and one HC participant). Figure 2 (Labeled as “PD9” and “HC17” in A). Based on comprehensive T-cell analysis, none of the three inherited PDs were identified as outliers compared to iPD in the PCA plot. Similar to the CyTOF data, no difference was observed in the frequency of total CD8 T cells in fresh peripheral blood mononuclear cells (PBMCs) in iPD and HC. Figure 5 A). Unlike CyTOF data, the inventors of this application did not observe any significant differences in the frequencies of total T cells and total CD4 T cells between the two groups. Figure 5 A). The unchanged frequency of total T cells revealed by the inventors' FCM analysis may be due to the exclusion of granulocytes from PBMCs, which constitute the majority of the whole blood immune population. Although the total frequency of the major T cell population remained statistically unchanged in the FCM analysis ( Figure 5 A), but PCA analysis revealed a clear T-cell fingerprint profile in iPD, reflecting variations in specific T-cell subtypes. Figure 2 A).

[0177] In the most significantly altered immune subsets in iPD (p<0.05, fold change >1.4), FCM analysis again revealed a strong increase in the frequency of TEMRA in total CD8 T cells. Figure 2 B and C) independently confirmed the CyTOF results obtained by the inventors of this application. CD8 TEMRA cells reexpressed CD45RA but lost the expression of CD45RO, CCR7, and CD27. By analyzing the expression of CCR7 in the combination of CD45RA and CD45RO, the inventors of this application were able to better distinguish CD8 subsets and clearly identify CD45RA in total CD8 T cells in iPD. + CD45RO - CCR7 - Cells (CD8 TEMRA follow a simplified gating strategy) increase ( Figure 2 C).

[0178] To more rigorously identify CD8 TEMRA, the inventors of this application subsequently included CD27 in a separate staining plate. This is consistent with the results described above based on a simplified gating strategy. Figure 2 C), CD45RO in iPD - CCR7 - CD27 - The frequency of effector CD8 T cells also increased. Figure 5 B). Based on the fact that the fraction of CD45RA / CD45RO double-negative (mean: ~5%) or double-positive (mean: ~5%) cells in CD8 T cells is extremely small. Figure 5 C), most CD45RO - CCR7 - CD27 - Effector CD8 T cells ( Figure 5 B) should be CD45RA + CCR7 - CD27 - CD8 TEMRA, hence also called CD8 TEMRA. Meanwhile, TCM (CD45RO) + CCR7 + CD27+ ) and transitional memory (TM) (CD45RO) + CCR7 - CD27 + CD8 T cell reduction ( Figure 5 D, E), while naive (CD45RO) - CCR7 + CD27 + No difference was observed in CD8 T cells between PD and HC. Figure 5 F). Decreased CD8 TCM and TM cells are associated with lower frequency of long-lived memory (KLRG1). - CD127 + CD8 T cells ( Figure 5 Consistent with G) and with CyTOF results. It is worth emphasizing that although the total CD8 cell count remained unchanged, the mean ratio between CD8 TEMRA and TCM increased significantly from 12.05 in HC to 42.90 in iPD. Figure 2 D). This ratio change indicates an imbalance in the differentiation process within iPD's CD8 T cells.

[0179] CD8 TEMRA alone, or the ratio of CD8 TEMRA to TCM, is a valuable peripheral cell index for the early diagnosis of iPD.

[0180] Given the substantial differences in CD8 TEMRA between iPD and HC in the inventors' cohort, they assessed the potential of using CD8 TEMRA as a peripheral cell diagnostic biomarker. The inventors first analyzed the potential correlation between the frequency of CD8 TEMRA and various available quantitative clinical information, such as age, disease duration, H&Y staging scale, UPDRS-III (Unified Parkinson's Disease Rating Scale III), LEDD (Levodopa Equivalent Daily Dose), and MOCA (Montreal Cognitive Assessment). Although no significant correlation was observed between CD8 TEMRA and most of these clinical data, it is noteworthy that the frequency of CD8 TEMRA in PD showed a correlation with the duration of disease from the onset of initial symptoms (…). Figure 3 A) and duration after clinical diagnosis ( Figure 3 A significant negative correlation was observed between B and CD8 TEMRA. This suggests that the CD8 TEMRA population may be more involved in the early stages of iPD than the later stages. Interestingly, based solely on the frequency of CD8 TEMRA, samples from early to mid-stage iPD may have been well distinguished from HC samples with an area under the curve (AUC) of 0.7662. Figure 3 C). Since the frequency of CD8 TEMRA is negatively correlated with disease duration, the inventors of this application applied an alternative receiver operating characteristic (ROC) analysis by focusing on patients diagnosed only within 5 years. Notably, based on the CD8 TEMRA frequency of these patients, an excellent AUC value of 0.8580 was obtained (for the definition of the AUC range, see Mandrekar, Receiver operating characteristic curve in diagnostic test assessment, 2010 Sep;5(9):1315-6. doi: 10.1097 / JTO.0b013e3181ec173d.2010; 100% sensitivity and 70.83% specificity at a cutoff of 40.3% of the percentage of CD8 TEMRA in CD8). Figure 3 D).

[0181] Since the frequencies of CD8 Treg (FOXP3+CD8 T cells) and ILC2 also show differences between iPD and HC, the inventors of this application assessed whether those subtypes could provide additional value by analyzing them together with CD8 TEMRA in the same plot. Figure 3 E, F). Since the ratio between CD8 TEMRA and CD8 Treg may represent the CD8 effector potential in a given individual, the inventors of this application also analyzed diagnostic values ​​by integrating the ratio and frequency of CD8 TEMRA. Figure 3 G). Interestingly, the frequency of CD8 Treg, the frequency of ILC2, and the ratio between CD8 TEMRA and CD8 Treg did not substantially increase the potential of CD8 TEMRA as a biomarker, always leaving six out of seven HC samples mixed in the 'PD region' ( Figure 3G). Surprisingly, detailed analysis of the graph showing the ratio between CD8 TEMRA and CD8 Treg versus the frequency of CD8 TEMRA revealed that six of the seven 'indistinguishable' HC samples were male. Noticing the sex bias, the inventors of this application performed another ROC analysis including only female samples. Very encouragingly, an outstanding AUC value of up to 0.9125 was achieved (the range of outstanding AUC values ​​can be described as in Mandrekar, Receiver operating characteristic curve in diagnostic test assessment, 2010 Sep;5(9):1315-6. doi: 10.1097 / JTO.0b013e3181ec173d.) Figure 3 H). When analyzing only female participants, ROC analysis using the ratio between CD8 TEMRA and CD8 Treg also showed excellent diagnostic value (H). Figure 3 I, J). With the significant increase in the CD8 TEMRA to CD8 TCM ratio in iPD, the inventors of this application also used this ratio for ROC analysis. Excitingly, even after analyzing all patients, an excellent AUC value of 0.8503 was obtained (…). Figure 3 K). Notably, outstanding AUC values ​​of 0.9470 or 0.9250 were achieved when analyzing patients diagnosed within the past five years or when analyzing only female patients. Figure 3 L, M). Even when analyzing only male patients and matched male HCs, an AUC value of 0.8120 was obtained ( Figure 3 In short, the inventors' data in this application strongly support the existence of CD8 TEMRA alone, or even better, the ratio between CD8 TEMRA and TCM, which may represent a previously unrecognized and reliably readily available cellular marker for the early diagnosis of iPD, particularly in female patients. Furthermore, the inventors' data in this application demonstrate that the ratio between CD8 TEMRA and TCM has a higher diagnostic value for both the overall patient population and subgroups of patients (e.g., those diagnosed within five years or female-only patients) than the ratio between CD8 TEMRA and Treg.

[0182] Validation in another cohort and enhanced cytotoxic pathways in CD8 cells

[0183] Given their high clinical diagnostic potential, the inventors of this application further validated key results regarding CD8 T cells in another subcohort (12 HC patients vs. 11 iPD patients) of their Luxembourg Parkinson's Study (Hipp et al., The Luxembourg Parkinson's Study: A Comprehensive Approach for Stratification and Early Diagnosis, Front Aging Neurosci. 2018;10: 326, 2018) using FCM analysis (Table 3). Consistent with their data in the initial discovery cohort, there were no significant changes in total CD8 T cells, either in total CD3 or in live cells. Figure 4 A). Although all samples in the validation cohort were cryopreserved, the frequency of CD8 TEMRA still increased relative to HC in early to mid-stage iPD, while the frequency of CD8 TCM decreased. Figure 4 B, C). It is worth noting that the ratio between CD8 TEMRA and TCM remains significantly higher in early to mid-stage iPD than in HC. Figure 4 (D). However, the ratio of cryopreserved samples to fresh blood samples is generally low, which may be due to the effects of cryopreservation, as we and others have demonstrated.

[0184] discuss

[0185] The work of the inventors of this application demonstrates that the ratio between CD8 TEMRA and TCM may be a valuable peripheral non-invasive biomarker for early diagnosis, particularly for female iPD.

[0186] The inventors of this application further provide exemplary thresholds (Tables 4-7) for the ratio between CD8 TEMRA and TCM as a function of their specificity and sensitivity, which allow diagnostic engineers or clinicians to select specific thresholds based on specific diagnostic or prognostic needs.

[0187] Materials and methods

[0188] Queue design

[0189] The inventors of this application complied with Luxembourg's ethical guidelines and obtained ethical approval from the Luxembourg National Research Ethics Committee (CNER) (CNER Approval No. 20140713-SU1; with two additional amended notices dated February 20, 2023 and April 12, 2023). Informed consent was obtained from each participant before they were recruited to the study by the local clinical team in Luxembourg. All study participants were recruited from the Luxembourg Parkinson's Study (a national, single-center, observational longitudinal study with parallel healthy controls). The overall selection process was conducted in... Figure 1Provided in [the document]. As a first step, the inventors of this application screened HC and iPD patients aged 60–70 years (excluding three hereditary patients: a PD patient aged 48 years with two rare variants (a pathogenic homozygous variant N409S in GBA and another non-pathogenic heterozygous rare variant A383T in PINK1); a PD case aged 55 years with a non-pathogenic heterozygous variant K13R in GBA; and a PD patient aged 45 years with a homozygous pathogenic variant L369P in PINK1). The inventors of this application narrowed the selection to early to mid-stage PD with a mean disease duration of 6.6 years after diagnosis. Since aging is a major risk factor for PD and aging significantly affects the immune system, the inventors of this application focused on a relatively narrow age window (60–70 years) in the PD and corresponding HC groups. The inventors of this application also excluded potential participants if they were diagnosed with any immune-related disease (e.g., diabetes, cancer, chronic inflammatory diseases, autoimmune diseases, and acute infections) or if they were currently being treated with immunosuppressive drugs. Following the initial round of exclusion, cytomegalovirus (CMV) serological status was further tested using serum samples stored in a biobank for 150 PD and 58 HC participants. CMV has been well-established to contribute to the immunoaging process. To make the immunoassay comparable at such advanced age, the inventors of this application invited only HC and PD participants for fresh blood sampling if they were seropositive against CMV IgG. The inventors of this application invited only CMV seropositive participants for technical and epidemiological reasons. Technically, the inventors of this application could not rule out the possibility that some CMV seropositive participants might become CMV seropositive during the time interval between previous serum sampling and the current fresh blood sampling. Secondly, based on large-scale CMV seropositive studies in adjacent regions, most older adults were found to be CMV seropositive. Selecting CMV seropositive individuals within this age range would further severely limit the availability of suitable samples. Therefore, the inventors of this application decided to first analyze a more representative group of participants in their sixties. Therefore, a total of 28 individuals with positive PD and 24 individuals with positive HC CMV serological responses consented to be included in the discovery cohort requiring additional blood sampling. This was done to account for the diurnal rhythms of immune cell migration within the body. All blood samples from participants were collected in the morning and processed within 6 hours.

[0190] As a first validation, the inventors of this application analyzed cryopreserved available PBMCs from another independent subcohort of the same Luxembourg Parkinson's study. Eleven iPDs and twelve age- and sex-matched HCs were selected according to the same inclusion / exclusion criteria as the discovery cohort. Eight of the eleven iPDs were male, and five of the twelve HCs were male. Notably, due to issues with the availability of sufficiently suitable samples, the inventors of this application included two CMV serologically negative participants who otherwise met all other selection criteria for the HC group in the validation cohort. Excluding these two samples from the analysis did not alter the inventors' conclusions. For scRNA sequencing (scRNA-seq), considering the inventors' observation of female bias in CD8 T cells and intergroup comparability, the inventors of this application selected only female CMV+ participants (five iPDs and four matched HCs).

[0191] Detection of anti-CMV IgG

[0192] CMV infection is widespread in the population, and seropositivity is positively correlated with age. Previous reports have suggested that CMV infection promotes immunosenescence. To exclude potential bias in the inventors' analysis due to differences in CMV status between PD and HC, the inventors of this application measured CMV serology in all potential study participants (including HC) who met the inclusion and exclusion criteria, as well as in only selected participants who were seropositive for CMV. Figure 1 ELISA was performed on plasma samples from previous follow-ups, which were stored in a local biobank (Integrated Biobank of Luxembourg). The inventors of this application used a human anti-cytomegalovirus IgG ELISA kit (Abcam, ab108724) and followed the manufacturer's instructions.

[0193] PBMC separation

[0194] PBMC separation was performed as described in Capelle et al., Standard Peripheral Blood Mononuclear Cell Cryopreservation Selectively Decreases Detection of Nine Clinically Relevant T Cell Markers, Immunohorizons (2021) 5 (8): 711–720. In short, blood samples were taken from each participant in the morning using 10-ml vacuum blood collection tubes (K2EDTA blood collection tubes, 367525, BD). Under RT, SepMate was used... TM -50 tubes (85450, Stemcell) and Lymphoprep TM Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood by centrifugation at 1200×g for 20 min using (07801, StemCell). Cells were then rinsed with FCM (flow cytometry) buffer (Ca-free). 2+ / Mg 2+ Wash three times with PBS + 2% heat-inactivated FBS and count with a CASY cell counter.

[0195] Multi-panel multicolor flow cytometry analysis

[0196] Similar multi-panel, multi-color (up to 18 colors) FCM analyses have been fully established and performed by the inventors of this application in other human-sample-based studies (Capelle et al., Combinatorial analysis reveals highly coordinated early-stage immune reactions that predict later antiviral immunity in mild COVID-19 patients, 2022 Apr 19; 3(4): 100600). For ease of understanding, the inventors of this application describe the main procedures again. For each study participant, 1 million fresh PBMCs were stained for each of the 5 staining panels. Prior to cell staining, the PBMCs were incubated at 4°C for 15 min with 50 μL of Brilliant staining buffer (BD, 563794) containing 2.5 μL of Fc blocking antibody (BD, 564765). 50 μL of a 2× concentrated surface antibody master mixture diluted in Brilliant staining buffer was added to the cell suspension and incubated at 4°C for 30 min (Table 2). After three washing steps with FCM buffer (300×g, 5 min, 4℃), stained PBMCs were fixed at RT for 60 min using the fixation reagent of the True-Nuclear Transcription Factor Buffer Set (Biolegend, 424401). After fixation, the cells were centrifuged (400×g, 5 min, 4℃), resuspended in 200 μL of FCM buffer, and incubated overnight at 4℃. The next day, the PBMCs were washed once with permeation buffer from the same kit and resuspended in permeation buffer containing 2.5 μL of Fc blocking antibody. After incubation for 10 min, the cells were centrifuged, and the cell pellet was resuspended in 100 μL of permeation buffer containing antibody for intracellular targets, and incubated at RT for 30–40 min. Finally, the cells were washed three times with permeation buffer and resuspended in 100 μL of FCM buffer for use in BD LSRFortessa. TMData were collected online. FlowJo v10 software was used for data analysis. Those skilled in the art will understand that pre-gating of live lymphocyte single-cell populations is necessary before setting gating to identify the specific CD8 subtypes mentioned herein (e.g., CD8 TEMRA, CD8 TCM). It is noteworthy that, using the inventors' assumption-free approach, we could not predict the CD8 TEMRA results. The inventors never used CD45RA, CCR7, and CD27 on the same panel, which is why they had to demonstrate CD8 TEMRA results by combining different gating strategies from different panels. The inventors wish to point out that in their cytometric analysis, they manually checked the gating for each immunosubtype from each donor and could slightly adjust the gating positions for different donors as needed.

[0197] Table 1. Mass cytometry (CyTOF) antibodies used for staining whole blood.

[0198]

[0199]

[0200] *Internal conjugation was performed using the Maxpar X8 Antibody Labeling Kit MDIPA (201325, Fluidigm). The predicted theoretical amounts used for each reaction of the internally conjugated Abs CD117-142Nd, KLRG1-159Tb, NKP44-162Dy, CD49d-169Tm, PD1-175Lu, and LAG3-165Ho were 0.5 μg, 0.17 μg, 0.5 μg, 0.05 μg, 0.05 μg, and 0.17 μg, respectively. Theoretical concentrations were calculated based on a projected average recovery of 60% for the antibody metal labeling procedure.

[0201] Table 2. PBMCs of participants analyzed in this study were stained using flow cytometry (FCM) antibodies.

[0202]

[0203]

[0204] * Different fluorescent dyes for the same marker may be used in different staining plates, since we used five staining plates in parallel.

[0205] Table 3. Antibodies used for intracellular cytotoxicity marker analysis via FCM and / or for CD8 subtype sorting via FACS.

[0206]

[0207] It is worth noting that for extracellular mixtures, the final volume for each reaction is 50 μL, diluted in Brilliant staining buffer. For intracellular mixtures, the final volume for each reaction is 100 μL, diluted in permeabilization buffer.

[0208] Table 4. CD8 TEMRA: CD8 TCM ratio: exemplary thresholds and corresponding sensitivity, specificity and likelihood ratios (all subjects).

[0209]

[0210]

[0211] Table 5. CD8 TEMRA: CD8 TCM ratio: exemplary thresholds and corresponding sensitivity, specificity and likelihood ratio (female subjects).

[0212]

[0213] Table 6. CD8 TEMRA: CD8 TCM ratio: exemplary thresholds and corresponding sensitivity, specificity and likelihood ratios (subjects clinically diagnosed within 5 years).

[0214]

[0215]

[0216] Table 7. CD8 TEMRA: CD8 TCM ratio: exemplary thresholds and corresponding sensitivity, specificity and likelihood ratio (male subjects).

[0217]

[0218]

Claims

1. An ex vivo method for diagnosing, prognosing, or monitoring Parkinson's disease (PD) in a subject, the method comprising: (a) Determine the amounts of CD8 TEMRA and CD8 TCM in biological samples from the subjects; and (b) Determine the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM) in the biological sample from the subject.

2. The method according to claim 1, wherein the biological sample is a body fluid sample, preferably a peripheral whole blood sample.

3. The method according to claim 1 or 2, wherein the biological sample comprises peripheral blood mononuclear cells (PBMCs).

4. The method according to any one of claims 1 to 3, comprising the following steps: (i) Determine the ratio of CD8 TEMRA to CD8 TCM in biological samples from the subjects; (ii) Compare the ratio of CD8 TEMRA to CD8 TCM determined in (i) with a reference; (iii) The ratio of CD8 TEMRA to CD8 TCM determined in (i) is found to be different from or not different from the reference value; and (iv) Attributing the findings, whether different or not, to a specific diagnosis or prognosis of the subject's PD.

5. The method according to claim 4, in, If the reference corresponds to the CD8 TEMRA to CD8 TCM ratio in a biological sample unaffected by PD, such as a healthy sample, then a significant increase in the CD8 TEMRA to CD8 TCM ratio determined in (i) compared to the reference allows for the diagnosis or prognosis of PD in the subject; or Wherein, if the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample affected by PD, then the lack of significant difference between the CD8 TEMRA to CD8 TCM ratio determined in (i) and the reference allows for the diagnosis or prognosis of PD in the subject.

6. The method according to any one of the preceding claims, wherein the step of determining the quantity of CD8 TEMRA and CD8 TCM is performed using cytometry or other single-cell characterization methods.

7. A kit, particularly a kit for diagnosing, prognosing, or monitoring PD in subjects, said kit comprising: (a) A tool specifically designed to determine the ratio of CD8 TEMRA to CD8 TCM in biological samples from subjects; and (b) A reference to the ratio, wherein the reference represents a value that allows for the diagnosis or prognosis of PD. (a) and (b) are the only biological reagents present in the kit.

8. The kit according to claim 7, wherein the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample unaffected by PD, such as a healthy sample, or wherein the reference corresponds to the ratio of CD8 TEMRA to CD8 TCM in a biological sample affected by PD.

9. The kit according to claim 7 or 8, wherein the tool specifically designed for determining the ratio of CD8 TEMRA to CD8 TCM comprises one or more binding agents that allow CD8 TEMRA and CD8 TCM to be distinguished from other CD8+ T cells.

10. The kit according to any one of claims 7 to 9 is used for the diagnosis, prognosis, or monitoring of PD based on the determination of the ratio of CD8 TEMRA to CD8 TCM in a subject's biological sample.

11. A computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, the computer program product including a computer-readable storage medium thereon having a computer program mechanism encoded thereon, wherein the computer program mechanism causes the computer to perform step (b) of the method of claim 1, step (b) comprising determining the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM) in the biological sample from the subject.

12. A computer-executed method for diagnosing, prognosing, or monitoring Parkinson's disease (PD) in a subject, the method comprising determining the ratio of CD8 terminally differentiated effector T cells (CD8 TEMRA) to CD8 central memory T cells (CD8 TCM) in a biological sample from the subject.

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