Reversing epigenetic T cell exhaustion

This 2019 worldwide discussion among 18 experts concerned T cell exhaustion:

“‘T cell exhaustion’ is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours.

Key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.”

There were nearly a dozen viewpoints on even “What do we mean by T cell exhaustion and/or dysfunction and how would you define this state?” 🙂

Answers to the question “What are the key controversies and outstanding research questions?” included:

  • “What are the cellular signalling and transcriptional pathways that drive the conversion to an exhausted T cell phenotype, and how can the chromatin and transcriptional changes of exhaustion be reversed in individual exhausted cells?
  • Whether and how we can manipulate signalling pathways to both activate and maintain T cell responses remain open questions, as does the question of whether pharmacological manipulations can reverse the epigenetic changes associated with exhaustion versus expand less-exhausted populations.
  • We need to define better the effects of the microenvironment on the induction of T cell exhaustion, the developmental trajectories of exhaustion and the point at which and extent to which exhaustion can be reversed. Understanding the consequences of unleashing T cells from exhaustion will also be crucial to designing the most effective therapeutic interventions.
  • When and how exhausted T cell populations are formed. The original view that they are terminally differentiated descendants of formerly ‘normal’ effector T cells has been challenged.
  • Whether the predysfunctional T cells themselves, or their more differentiated (and phenotypically dysfunctional) progeny, form the ultimate effector pool for control of human tumours.
  • How do the functions and states (subpopulations) of exhausted T cells change over time? Can the epigenetic state of exhaustion be reversed to form true effector or memory T cells, and is this required for improved cancer immunotherapy?
  • There is no definitive marker for exhausted T cells, although TOX may prove to be useful. Transcriptional profiles are informative, but epigenetic changes are more specific and robust. A major clinical question is whether exhausted T cells can be, or indeed need to be, reprogrammed to achieve therapeutic benefit.” “Defining ‘T cell exhaustion'” (not freely available)


Transgenerational epigenetic inheritance of thyroid hormone sensitivity

My 500th curation is a 2019 Portuguese human study of Azorean islanders:

“This study demonstrates a transgenerational epigenetic inheritance in humans produced by exposure to high TH [thyroid hormone] in fetal life, in the absence of maternal influences secondary to thyrotoxicosis. The inheritance is along the male line.

The present work took advantage of the relatively frequent occurrence of fetal exposure to high TH levels in the Azorean island of São Miguel. This is the consequence of a missense mutation in the THRB gene causing the amino-acid replacement R243Q, resulting in reduced affinity of the TH receptor beta (TRβ) for TH and thus RTHβ.

Its origin has been traced to a couple who lived at the end of the 19th century. F0 represented the third generation and F3 the sixth and seventh generation descendant.”

The study added evidence for human transgenerational epigenetic inheritance. However, the lead sentence in its Abstract wasn’t correct:

“Evidence for transgenerational epigenetic inheritance in humans is still controversial, given the requirement to demonstrate persistence of the phenotype across three generations.”

Although found in this study, there is no “requirement to demonstrate persistence of the phenotype.” Observing the same phenotype in each generation is NOT required for human transgenerational epigenetic inheritance to exist!

Animal transgenerational studies have shown that epigenetic inheritance mechanisms may both express different phenotypes for each generation:

and entirely skip a phenotype in one or more generations!

  • Transgenerational pathological traits induced by prenatal immune activation found a F2 and F3 generation phenotype of impaired sociability, abnormal fear expression and behavioral despair – effects that weren’t present in the F1 offspring;
  • The transgenerational impact of Roundup exposure “Found negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology. In contrast, dramatic increases in pathologies in the F2 generation grand-offspring, and F3 transgenerational great-grand-offspring were observed.” (a disease phenotype similarly skipped the first offspring generation);
  • Epigenetic transgenerational inheritance mechanisms that lead to prostate disease “There was also no increase in prostate histopathology in the directly exposed F1 or F2 generation.” (a prostate disease phenotype skipped the first two male offspring generations before it was observed in the F3 male offspring); and
  • Epigenetic transgenerational inheritance of ovarian disease “There was no increase in ovarian disease in direct fetal exposed F1 or germline exposed F2 generation. The F3 generation can have disease while the F1 and F2 generations do not, due to this difference in the molecular mechanisms involved.” (an ovarian disease phenotype similarly skipped the first two female offspring generations before it was observed in the F3 female offspring).

Details of epigenetic inheritance mechanisms were provided in Another important transgenerational epigenetic inheritance study. Mechanisms from fetal exposure to the fungicide vinclozolin were compared with mechanisms from fetal DDT exposure, and summarized as:

The fetal exposure initiates a developmental cascade of aberrant epigenetic programming, and does NOT simply induce a specific number of DMRs [DNA methylation regions] that are maintained throughout development.

I emailed references to the studies in the first five above curations to the current study’s corresponding coauthor. They replied “What is the mechanism for the transgenerational inheritance you describe?” and my reply included a link to the sixth curation’s study.

Are there still other transgenerational epigenetically inherited effects due to fetal exposure to high thyroid hormone levels? “Reduced Sensitivity to Thyroid Hormone as a Transgenerational Epigenetic Marker Transmitted Along the Human Male Line”

Reversal of aging and immunosenescent trends

The title of this post is essentially the same as the 2019 human clinical trial:

“Epigenetic aging can be reversed in humans. Using a protocol intended to regenerate the thymus, we observed protective immunological changes, improved risk indices for many age‐related diseases, and a mean epigenetic age approximately 1.5 years less than baseline after 1 year of treatment.

This is to our knowledge the first report of an increase, based on an epigenetic age estimator, in predicted human lifespan by means of a currently accessible aging intervention.”

“Example of treatment‐induced change in thymic MRI appearance. Darkening corresponds to replacement of fat with nonadipose tissue. White lines denote the thymic boundary. Volunteer 2 at 0 (a) and 9 (b) months” “Reversal of epigenetic aging and immunosenescent trends in humans”

Here’s a 2017 interview with the clinical trial lead author:

“You might also say that what also happened was to just postpone death from infectious diseases to after 60-65 years of age, which means that the same basic problem still remains.”

The popular press botched the facts as they usually do. I won’t link the UK Independent article because they couldn’t be bothered to even define epigenetic clock correctly.

A science journal article did a better job of explaining the study to readers. However, they often used hyperbole instead of trying to promote understanding.

Josh Mitteldorf’s blog post 1st Age Reversal Results—Is it HGH or Something Else? provided the most informative explanations:

“In 2015, Fahy finally had funding and regulatory approval to replicate his one-man trial in a still-tiny sample of ten men, aged 51-65. That it took so long is an indictment of everything about the way aging research is funded in this country; and not just aging – all medical research is prioritized according to projected profits rather than projected health benefits.”

Take care reading the post’s comments. Both non-scientist (such as Mark, Adrian, and others) and scientist commentators (such as Gustavo, Jeff, and others) attempted to hijack the discussion into their pet theories of reality in which they imagined themselves to be the definitive authorities. My discussion comment – with respect to a Mayo Clinic warning about DHEA – was: “19 instances of the word ‘might’ doesn’t lend itself to credibility.”

PNAS politics in the name of science

This 2019 Germany/Canada human fetal cell study was a Proceedings of the National Academy of Sciences of the United States of America direct submission:

“In a human hippocampal progenitor cell line, we assessed the short- and long-term effects of GC [glucocorticoid] exposure during neurogenesis on messenger RNA expression and DNA methylation profiles. Our data suggest that early exposure to GCs can change the set point of future transcriptional responses to stress by inducing lasting DNAm changes.”

The study’s basic finding was that cells had initial responses to stressors that primed them for subsequent stressors. Since this finding wasn’t new, the researchers tried to make it exciting by applying it to novel contexts that were yet circumscribed by official paradigms.

Hypothesis-seeking associations of human fetal hippocampal cell behaviors with human behaviors were flimsy stretches, as were correlations to placental measurements. These appeared to have been efforts to find headline-making effects.

There wasn’t even a hint of the principle described in Epigenetic variations in metabolism:

“Because of the extreme interconnectivity of cell regulatory networks, even at the cellular level, predicting the impact of a sequence variant is difficult as the resultant variation acts:

  • In the context of all other variants and
  • Their potential additive, synergistic and antagonistic interactions.

This phenomenon is known as epistasis.”

It would have condemned pet models of reality to admit that a cell exists in multiple contexts of other cells with potential additive, synergistic, and antagonistic interactions.

A research proposal to trace a specific cell type’s behaviors – while isolated from their extremely interconnected networks – to trillion-celled human behaviors would be rejected in less-politicized organizations.

Sanctioned speculations manifested in this paper with phrases such as “although not significant..” and “although not directly tested..” The study’s title was probably a disappointment in that it conformed to the study’s evidence.

Involvements of psychiatry departments at the pictured Kings College, Harvard, etc., as part of PNAS entrenched politics, retard advancements of science past approved paradigms.

This is my final curation of PNAS papers. “Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation”

Developmental disorders and the epigenetic clock

This 2019 UK/Canada/Germany human study investigated thirteen developmental disorders to identify genes that changed aspects of the epigenetic clock:

“Sotos syndrome accelerates epigenetic aging [+7.64 years]. Sotos syndrome is caused by loss-of-function mutations in the NSD1 gene, which encodes a histone H3 lysine 36 (H3K36) methyltransferase.

This leads to a phenotype which can include:

  • Prenatal and postnatal overgrowth,
  • Facial gestalt,
  • Advanced bone age,
  • Developmental delay,
  • Higher cancer predisposition, and, in some cases,
  • Heart defects.

Many of these characteristics could be interpreted as aging-like, identifying Sotos syndrome as a potential human model of accelerated physiological aging.

This research will shed some light on the different processes that erode the human epigenetic landscape during aging and provide a new hypothesis about the mechanisms behind the epigenetic aging clock.”

“Proposed model that highlights the role of H3K36 methylation maintenance on epigenetic aging:

  • The H3K36me2/3 mark allows recruiting de novo DNA methyltransferases DNMT3A (in green) and DNMT3B (not shown).
  • DNA methylation valleys (DMVs) are conserved genomic regions that are normally found hypomethylated.
  • During aging, the H3K36 methylation machinery could become less efficient at maintaining the H3K36me2/3 landscape.
  • This would lead to a relocation of de novo DNA methyltransferases from their original genomic reservoirs (which would become hypomethylated) to other non-specific regions such as DMVs (which would become hypermethylated and potentially lose their normal boundaries),
  • With functional consequences for the tissues.”

The researchers improved methodologies of several techniques:

  1. “Previous attempts to account for technical variation have used the first 5 principal components estimated directly from the DNA methylation data. However, this approach potentially removes meaningful biological variation. For the first time, we have shown that it is possible to use the control probes from the 450K array to readily correct for batch effects in the context of the epigenetic clock, which reduces the error associated with the predictions and decreases the likelihood of reporting a false positive.
  2. We have confirmed the suspicion that Horvath’s model underestimates epigenetic age for older ages and assessed the impact of this bias in the screen for epigenetic age acceleration.
  3. Because of the way that the Horvath epigenetic clock was trained, it is likely that its constituent 353 CpG sites are a low-dimensional representation of the different genome-wide processes that are eroding the epigenome with age. Our analysis has shown that these 353 CpG sites are characterized by a higher Shannon entropy when compared with the rest of the genome, which is dramatically decreased in the case of Sotos patients.” “Screening for genes that accelerate the epigenetic aging clock in humans reveals a role for the H3K36 methyltransferase NSD1”

Caloric restriction’s epigenetic effects

This 2019 US review subject was caloric restriction (CR) without malnutrition:

“Cellular adaptation that occurs in response to dietary patterns can be explained by alterations in epigenetic mechanisms such as DNA methylation, histone modifications, and microRNA. Epigenetic reprogramming of the underlying chronic low-grade inflammation by CR can lead to immuno-metabolic adaptations that enhance quality of life, extend lifespan, and delay chronic disease onset.

Short- and long-term CRs produce significant changes in different tissues and across species, in some animal models even with sex-specific effects. Early CR onset may cause a different and even an opposite effect on physiological outcomes in animal models such as body weight.”


1. Charts usually don’t have two different values plotted on the same axis. There wasn’t evidence that equated survival with methylation drift per the above graphic. Methylation drift should point in the opposite direction of survival, if anything.

2. No mention was made of the epigenetic clock method of measuring age acceleration, although it’s been available since 2013 and recent diet studies have used it. The sole citation of an age acceleration study was from 2001, which was unacceptable for a review published in 2019.

3. The review provided many cellular-level details about the subject. However, organism-level areas weren’t sufficiently evidenced:

A. Arguments for an effect usually include explanations for no effect as well as opposite effects. The reviewers didn’t provide direct evidence for why, if caloric restriction extended lifespan, caloric overabundance produced shorter lifespans.

B. Caloric restriction evidence was presented as if only it was responsible for organism-level effects. Other mechanisms may have been involved.

An example of such a mechanism was demonstrated in a 2007 rodent study Reduced Oxidant Stress and Extended Lifespan in Mice Exposed to a Low Glycotoxin Diet which compared two 40%-calorie-restricted diets.

The calories and composition of both diets were identical. However, advanced glycation end product (AGE) levels were doubled in standard chow because heating temperatures were “sufficiently high to inadvertently cause standard mouse chow to be rich in oxidant AGEs.”

The study found that a diet with lower chow heating temperatures increased lifespan and health span irrespective of caloric restriction!

  • The low-AGE calorie-restricted diet group lived an average of 15% longer (>20 human equivalent years) than the CR group.
  • 40% of the low-AGE calorie-restricted diet group were still alive when the last CR group member died.
  • The CR group also had significantly more: 1) oxidative stress damage; 2) glucose and insulin metabolism problems; and 3) kidney, spleen, and liver injuries. “Epigenetic Regulation of Metabolism and Inflammation by Calorie Restriction” (not freely available)

A drug that countered effects of a traumatizing mother

This 2019 US rodent study concerned transmitting poor maternal care to the next generation:

“The quality of parental care received during development profoundly influences an individual’s phenotype, including that of maternal behavior. Infant experiences with a caregiver have lifelong behavioral consequences.

Maternal behavior is a complex behavior requiring the recruitment of multiple brain regions including the nucleus accumbens, bed nucleus of the stria terminalis, ventral tegmental area, prefrontal cortex, amygdala, and medial preoptic area. Dysregulation within this circuitry can lead to altered or impaired maternal responsiveness.

We administered zebularine, a drug known to alter DNA methylation, to dams exposed during infancy to the scarcity-adversity model of low nesting resources, and then characterized the quality of their care towards their offspring.

  1. We replicate that dams with a history of maltreatment mistreat their own offspring.
  2. We show that maltreated-dams treated with zebularine exhibit lower levels of adverse care toward their offspring.
  3. We show that administration of zebularine in control dams (history of nurturing care) enhances levels of adverse care.
  4. We show altered methylation and gene expression in maltreated dams normalized by zebularine.

These findings lend support to the hypothesis that epigenetic alterations resulting from maltreatment causally relate to behavioral outcomes.”

“Maternal behavior is an intergenerational behavior. It is important to establish the neurobiological underpinnings of aberrant maternal behavior and explore treatments that can improve maternal behavior to prevent the perpetuation of poor maternal care across generations.”

The study authors demonstrated intergenerational epigenetic effects, and missed an opportunity to also investigate transgenerational epigenetically inherited effects. They cited reference 60 for the first part of the above quotation, but that reviewer misused the transgenerational term by applying it to grand-offspring instead of the great-grand-offspring.

There were resources available to replicate the study authors’ previous findings, which didn’t show anything new. Why not use such resources to uncover evidence even more applicable to humans by extending experiments to great-grand-offspring that have no potential germline exposure to the initial damaging cause?

Could a study design similar to A limited study of parental transmission of anxiety/stress-reactive traits have been integrated? That study’s thorough removal of parental behavior would be an outstanding methodology to confirm by falsifiability whether parental behavior is both an intergenerational and a transgenerational epigenetic inheritance mechanism.

Rodent great-grand-offspring can be studied in < 9 months. It takes > 50 years for human studies to reach the transgenerational generation. Why not attempt to “prevent the perpetuation of poor maternal care across generations?”

Isn’t it a plausible hypothesis that humans “with a history of maltreatment mistreat their own offspring?” Isn’t it worth the extra effort to extend animal research to investigate this unfortunate chain? “Pharmacological manipulation of DNA methylation normalizes maternal behavior, DNA methylation, and gene expression in dams with a history of maltreatment”