Art Courtesy of Lynn Dai.
Sometimes, our immune cells stage a mutiny, turning their defensive weapons against the body they’re meant to protect. This betrayal is the defining feature of autoimmune disorders: the immune system, which typically protects us from germs and foreign substances, begins to mistake our own cells as foreign threats—so it attacks. One such disorder is multiple sclerosis (MS). In MS, T cells, a type of immune cell, misidentify parts of the brain as foreign and attack their myelin sheaths, an insulating fatty lining that surrounds neurons. Without these sheaths, neural signaling slows dramatically, leading to a wide range of symptoms, from visual problems and muscle weakness to urinary dysfunction.
Normally, conventional T cell activity indicates a robust immune system, but it must be kept in check to prevent overreaction. This job falls to a specialized group of T cells called regulatory T cells (Tregs), which are responsible for distinguishing between self and non-self cells and suppressing overactive conventional T cell responses. While Tregs may not be as celebrated as their conventional counterparts, they are crucial players in the immune system. They were first characterized in mouse models in 1996 by Shimon Sakaguchi, a professor at Osaka University, while their role in humans was described in 2001 by David Hafler, now the William S. and Lois Stiles Edgerly Professor of Neurology and Immunology at the Yale School of Medicine. In MS, the prevailing hypothesis is that when Tregs malfunction, T cell activity goes unchecked, and autoimmunity ensues.
The Hafler Lab has focused on studying Tregs to better understand the basis of MS. In a recent study published in Science Translational Medicine, they examined the mechanism behind regulatory T cell dysfunction. “We’ve spent the past quarter century looking at why [Tregs] are defective, but this paper really worked out the molecular mechanism for loss-of-function,” said Hafler, the senior author of the study.
Identifying A “Master Switch” in MS
The team employed a multi-omics approach, which integrates multiple types of biological data, including gene expression and gene regulation. They isolated regulatory T cells from MS patients and healthy individuals as controls, and then performed RNA sequencing on these cells. RNA sequencing allows researchers to quantify the expression of genes in cells of interest, providing insight into which genes are active. While DNA stores the genetic information needed for making proteins, gene expression first involves transcribing DNA into RNA, which serves as the template for proteins. Although DNA sequences are identical across almost all cells in the body, the expression levels of different genes within our DNA differ greatly. Since proteins drive most biochemical reactions in cells, fluctuations in gene expression levels can lead to changes in cell function.
“Studies have been focused on how specific molecules cause Treg dysfunction in MS, but we’ve never explored unbiased gene expression profiles from MS patient Tregs,” said Tomokazu Sumida, an assistant professor of neurology at Yale School of Medicine and first author of this study.
When Sumida and colleagues compared the RNA-sequencing data of MS individuals to that of the controls, they identified genes with differing expression levels between the two groups. One gene was of special interest: PRDM1.
“When you look at the differential display, [PRDM1] was one of the highest—it was strikingly high. And the other factors that were high were RNAs either induced or suppressed by PRDM1,” Hafler said. He explained that PRDM1 encodes B Lymphocyte-Induced Maturation Protein 1 (BLIMP1), a transcription factor that helps regulate the transcription of other genes. PRDM1 expression is increased in MS patients; it follows that the expression levels of genes that it up- or down-regulates should also deviate from controls. For example, ID3, a gene repressed by BLIMP1, was among the most significantly under-expressed genes in MS Tregs.
To confirm that the PRDM1 overexpression was consistent across different subtypes of Tregs, the researchers also performed single-cell RNA sequencing. Their results showed that PRDM1 was indeed consistently overexpressed across all Tregs.
Short Form PRDM1: Why Mouse Models Fall Short
PRDM1 is relatively well-characterized in mouse models of MS, where its higher expression is usually linked to less severe disease. Researchers were therefore surprised to find PRDM1 was overexpressed in humans with MS. “We were very confused because there are very nice studies which showed that PRDM1 in animal models of MS is associated with increased Treg function and less disease,” Hafler said.
Clinical samples were the missing link to solving this discrepancy between humans and mice. It turns out that PRDM1 exists in two forms in humans: the full-length form (PRDM1-L) and a shorter form, PRDM1-S, which exists only in dry-nosed mammals, such as primates and rodents. PRDM1-S encodes a truncated form of BLIMP1 named BLIMP1-S, which inhibits the normal function of full-length BLIMP1. While this has been studied in immunology, it was primarily in the context of cancer, not autoimmunity.
The researchers measured PRDM1-S and PRDM1-L expression in different immune cells of healthy participants and found that their proportions varied across cell types. They went on to characterize PRDM1 expression in MS patients. Additional analysis of RNA-sequencing of Tregs indicated that while PRDM1-S expression was considerably elevated in patients with MS, the expression of PRDM1-L also increased—though not to a statistically significant degree—which maintained a constant ratio of short- to long-form PRDM1. This suggests that the effects seen in MS are not merely due to a relative decrease in PRDM1-L activity, as the relative amounts of both forms remain unchanged. Still, PRDM1-S upregulation was correlated with lower expression of proteins that play an important role in Treg suppression of conventional T cells. It was therefore hypothesized that PRDM1-S must have an independent effect in MS, separate from its potential interaction with PRDM1-L.
Armed with this information, the researchers looked for downstream pathways that were most affected by PRDM1-S. A combination of biochemical and RNA-sequencing analyses revealed that PRDM1-S overexpression induced the expression of a gene called SGK1, named after the protein that it encodes, serum and glucocorticoid-regulated kinase 1. This was an interesting finding because the relationship between PRDM1 and SGK1 has been previously implicated in numerous autoimmune diseases, including allergies and inflammatory bowel disease. The final linchpin came by identifying the effects of SGK1 in suppressing the expression and stabilization of the protein FOXP3, a protein crucial for Treg function. FOXP3 instability is a known factor in Treg dysfunction in MS.
Putting the pieces together for this pathway explained some patient data that had long puzzled physicians. MS treatments have included low-salt diets, based on the observation that salt exacerbates autoimmune flare-ups. Now, researchers know that a high-salt environment leads to increased PRDM1-S expression. Understanding that the PRDM1-S/SGK1 pathway drives MS patient symptoms underscores the importance of studying this pathway for the development of future therapeutics.
Transcription Factors for the Transcription Factor?
The researchers also sought to understand the upstream causes of PRDM1-S upregulation in Tregs, such as whether certain transcription factors regulate PRDM1-S and PRDM1-L differently. They performed an assay to find regions of DNA that are more loosely packed, which corresponds to increased accessibility for transcription factor binding and therefore increased rates of transcription. In the case of PRDM1, overall accessibility was comparable between MS patients and controls.
Using this data, it is also possible to identify the binding sites, also known as “footprints,” of specific factors. This analysis reveals the extent of transcription factor interaction with a DNA sequence based on its chemistry rather than how tightly the DNA is packed. Here, the researchers found increased binding of activating protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factor families in MS patients. These factors have previously been associated with immune cell differentiation that gives rise to Tregs. The team inhibited the expression of BATF and IRF4 transcription factors, members of the AP-1 and IRF families, respectively, and found that knockdown of either transcription factor led to higher PRDM1-S expression. Thus, losing these two key Treg regulators seems to be a potential cause for aberrant PRDM1-S induction in MS.
Broad Implications and Future Directions
“I think PRDM1-S is a highly overlooked transcription factor in humans,” Sumida said. Different immune cell types show distinct expression patterns of the short and long forms of PRDM1. For example, B cells, which produce antibodies in adaptive immune response, are tightly regulated to express only PRDM1-L, and upregulation of PRDM1-S is associated with B cell lymphoma. On the other hand, in natural killer cells, PRDM1-S expression is much higher than PRDM1-L expression. The Sidi Chen Lab at Yale recently reported that a CRISPR-generated shortened version of PRDM1 led to higher CAR-T (chimeric antigen receptor T-cell therapy) efficacy in cancers.Given the substantial implications of PRDM1-S, the Hafler and Sumida Labs plan to continue their research on this transcription factor. “We’re working with some structural biologists in crystallography to come up with small molecules that block short-form PRDM1,” Hafler said. Since the transcription factor is involved in many cellular pathways and autoimmune disorders, they hope that it will be a particularly lucrative molecular target for drug development. Eventually, Hafler is interested in targeting SGK as well, since it has a direct relationship with the physiological manifestations of MS. As the team works on elucidating the effects of PRDM1-S and its downstream effects, their findings may pave the way for novel treatments for MS and other autoimmune disorders.