Individual Autoantibody Profiles: The Effect of Physiological Stressors on Profile Fidelity

Rahil Kheirkhah, Ph.D1, Robert Nagele, Ph.D.11. Rowan University School of Osteopathic Medicine, Stratford, NJ

Proposed Function of Autoantibodies

Introduction Pregnancy as a Stressors and aAB profiles

Human Protein Microarrays

It is a fact historically accepted in the field of immunology that the presence of autoantibodies(aABs) in the blood is a relatively rare event linked to some type of pathology, such as autoimmunedisorders. More current research, however, now challenges the idea of central tolerance, withevidence supporting the presence of thousands of self-reactive aABs in the blood, even in theabsence of pathology. This idea supports a homeostatic role for this subset of the immune system,including the response to sterile injury, immunosurveillance of cancer, facilitating wound resolutionand tissue regrowth, and cell and tissue debris clearance.

The nature of the response of these aABs to pathology has led to the exploration of their utility asbiomarkers for a number of diseases, including but not limited to Mild Cognitive Impairment,Alzheimer’s Disease and Parkinson’s Disease. Additionally, the ubiquitous presence of suchcomplex aAB profiles in the blood has triggered a new field of endeavor, with the goal ofelucidating details of the role of such aABs in the maintenance of body-wide homeostasis, as wellas in the body in response to stressors.

Autoantibody Fingerprints

References

The figure to the right is a comparison of aAB reactivity in plasma and CSF among four different individuals. RelaiveFluorescence Units (RFUs) from an identical block of 75 randomly selected proteins were plotted for four individual subjects to demonstrate the close matching of plasma (red line) and CSF (blue line) aAB profiles. Dashed lines indicate peaks representing aABs that are common among the four individuals. Many of the aABs are still expressed, but the degree to

which these aABs are expressed is unique to each individual, giving rise to what we call an “aAB fingerprint.”

Considering the thousands of aAB in each person, no two people have the same aAB profile

1. Avrameas, S., Alexopoulos, H., & Moutsopoulos, H. M. (2018). Natural Autoantibodies: An Undersugn Hero of the Immune System and Autoimmune Disorders—A Point of View. Frontiers in Immunology, 9, 1320–1325. https://doi.org/10.3389/fimmu.2018.01320

2. Cohen, I. R. (2007). Biomarkers, self-antigens and the immunological homunculus. Journal of Autoimmunity, 29(4), 246–249. https://doi.org/10.1016/j.jaut.2007.07.0163. DeMarshall, C. A., Han, M., Nagele, E. P., Sarkar, A., Acharya, N. K., Godsey, G., Goldwaser, E. L., Kosciuk, M., Thayasivam, U., Belinka, B., & Nagele, R. G. (2015). Potential utility of autoantibodies as blood-based biomarkers for early

detection and diagnosis of Parkinson’s disease. Immunology Letters, 168(1), 80–88. https://doi.org/10.1016/j.imlet.2015.09.0104. DeMarshall, C. A., Nagele, E. P., Sarkar, A., Acharya, N. K., Godsey, G., Goldwaser, E. L., Kosciuk, M., Thayasivam, U., Han, M., Belinka, B., & Nagele, R. G. (2016). Detection of Alzheimer’s disease at mild cognitive impairment and disease

progression using autoantibodies as blood‐based biomarkers. Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring, 3(1), 51–62. https://doi.org/10.1016/j.dadm.2016.03.0025. DeMarshall, C., Goldwaser, E. L., Sarkar, A., Godsey, G. A., Acharya, N. K., Thayasivam, U., Belinka, B. A., & Nagele, R. G. (2017). Autoantibodies as diagnostic biomarkers for the detection and subtyping of multiple sclerosis. Journal of

Neuroimmunology, 309, 51–57. https://doi.org/10.1016/j.jneuroim.2017.05.0106. DeMarshall, C., Thayasivam, U., & Nagele, R. G. (2017). [P3-215]: UTILITY OF DISEASE-LINKED DEPLETION OF AUTOANTIBODIES AS BIOMARKERS FOR EARLY, BLOOD-BASED DETECTION AND MONITORING OF ALZHEIMER’s

AND OTHER NEURODEGENERATIVE DISEASES. Alzheimer’s & Dementia, 13(7S_Part_21), P1019–P1020. https://doi.org/10.1016/j.jalz.2017.06.14287. Kheirkhah, R., DeMarshall, C., Sieber, F., Oh, E., & Nagele, R. G. (2020). The origin and nature of the complex autoantibody profile in cerebrospinal fluid. Brain, Behavior, & Immunity - Health, 2, 100032.

https://doi.org/10.1016/j.bbih.2019.1000328. Levin, E. C., Acharya, N. K., Han, M., Zavareh, S. B., Sedeyn, J. C., Venkataraman, V., & Nagele, R. G. (2010). Brain-reactive autoantibodies are nearly ubiquitous in human sera and may be linked to pathology in the context of blood–brain

barrier breakdown. Brain Research, 1345, 221–232. https://doi.org/10.1016/j.brainres.2010.05.0389. Nagele, E. P., Han, M., Acharya, N. K., DeMarshall, C., Kosciuk, M. C., & Nagele, R. G. (2013). Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera, and Their Number Is Influenced By Age, Gender, and Disease. PLoS

ONE, 8(4), e60726. https://doi.org/10.1371/journal.pone.006072610. Norris, G. T., & Kipnis, J. (2018). Immune cells and CNS physiology: Microglia and beyond. Journal of Experimental Medicine, 216(1), 60–70. https://doi.org/10.1084/jem.20180199

Normal “wear and tear” in otherwise healthyindividuals generates soluble cellular debris. Thisdebris eventually makes its way into various bodyfluids, especially the blood. This cell-derived debriscreates a constant need for an extremely complexaAB profile to facilitate its daily removal andmaintaining a body-wide homeostasis via thecell/tissue debris clearance. This figure shows the fidelity of aAB profiles in a single individual

over a period of 9 years. Four different blocks of 50 randomlyselected proteins were probed with diluted plasma samples takenyears apart. aAB profiles remained essentially unchanged overthe 9 year period, providing strong evidence that, in the absenceof pathology, each individual has a unique and stable baselineaAB profile in the blood that demonstrates a high degree of fidelityover time.

In the absence of any pathology or stressors to the system, the aAB profile is maintained, indicating the presence of a stable baseline profileStressors include but are not limited to life events such as: Surgery, Infection, Pregnancy, Sudden Weight loss, Short/Long-term pathologies.

Examination of aAB profiles suggests that each individual possesses a unique aAB profile

Autoantibody Profile Fidelity

Longitudinal serum samples were collected from afemale patient before, during, and after her firstpregnancy. The figure to the right shows theprogression of the change from her baseline aAB toher profiles during pregnancy and then again, herprofile post-pregnancy. RFUs from four differentblocks of 50 randomly selected proteins are plotted.

There is a global drop in the number of aABsdetected in maternal serum during pregnancy, butthe number is later restored to pre-pregnant levels.

In as early as 7 months post-pregnancy, the aABprofile tend to restore to the levels of their baselineprofile. This supports the hypothesis that ourbaseline profiles adhere to a “set-point” and makean attempt to restore it.

Baseline aAB profiles adhere to a “set-point” and attempt to restore it.

Why is there a global reduction in aAB titers?1. Increased production of maternal cellular debris during gestation increases the demand for

aABs to clear this debris from the blood.2. During early gestation, organogenesis dominates and this remodeling produces an

abundance of cell and tissue debris (fetal origin), thus taxing the aAB debris-clearance systemand resulting in lower aAB titers.

3. Trans-placental transfer of maternal IgG to the fetus to establish the fetus’s own autoimmunebaseline profile at birth. This is necessary since newborn babies have no functional immunesystem and rely on the mother’s contribution.

Nitrocellulose-coated glass slide Human proteins spotted in duplicate

at defined addresses Proteins expressed in insect cells –

produce proteins with folding andpost-transcriptional modificationssimilar to mammalian cells

Each array contains nearly 10,000unique human proteins

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/protein-microarrays/technical-resources.html

Blood aAB profiles taken from a single individual, show a remarkable degree of fidelity over time.