Sarah Pizzano
Abstract
Human immunodeficiency virus (HIV), like many chronic infections, can cause the exhaustion of CD8+ T-cells. This disruption of normal T-cell function, including decreased cell proliferation and cytokine production, limits the immune system’s ability to eliminate infected cells. Although the PD-1 inhibitory pathway and various regulatory proteins have been implicated in maintenance of exhaustion, recent research on epigenetic contributors may offer an explanation as to how cells reach this state of declined function. Specifically, the shortening of telomeres and the regulation of transcription through methylation may contribute to many of the in functional losses in exhausted HIV-specific CD8+ T cells.
Introduction
During an acute infection antigen specific CD8+ T-cells respond to the expression of an antigen by differentiating into cytotoxic effector T-cells (1); Receptors on effector cells signal the T-cell to release various cytokines in order to induce apoptosis or viral inhibition in the targeted antigen-expressing cell (2). CD8+ T-cells play a key role in the adaptive immune system by differentiating further when exposed to antigens. In their further differentiated form these cells express antigen-specific receptors allowing faster recognition of the antigen and targeting of infected cells (1).
However, T-cell exhaustion occurs in cases of chronic infections and diseases including Hepatitis B, Cancer, and HIV (3). T-cell exhaustion is characterized by the functional impairment of the cell’s ability to express cytokines, cause cell death, and proliferate (3). In human immunodeficiency virus (HIV) induced exhaustion, cells exhibit increased expression of PD-1 inhibitory receptors, discussed later, and shortened telomeres in comparison to normal functioning T-cells (3). As a result exhausted cells fail to target and eliminate infected cells allowing for continued chronic infection (1). While other types of T-cells, such as CD4+ T-cells (helper cells) also experience exhaustion (2), this review will focus on CD8+ T-cells due to their therapeutic potential for HIV. Therapies attempting to reactivate exhausted CD8+ T-cells offer potential cures to HIV. Such treatments are difficult to create as the mechanisms responsible for T-cell exhaustion are not fully understood. Part of this lack of information is due to variability in mechanisms of exhaustion dependent on the type of chronic infection present (3). This review will consider HIV specific epigenetic mechanisms.
A variety of mechanisms exist regulating exhaustion, including intercellular signaling and protein pathways. Recently epigenetic factors contributing to transcriptional regulation and telomere length regulation have come into scrutiny as contributors to exhaustion. Therapies countering these epigenetic contributors in HIV infections may allow for the reactivation of T-cells and specific elimination of HIV infected cells by the immune system (4).
Transcriptional modification through methylation
PD-1 pathway PD-1 is an inhibitory cell surface receptor expressed on T-cells which regulates cytokine production and other T-cell functions (1). When T-cell receptors and antigen-specific CD8+ co-receptors interact with an antigen on an infected cell they activate the pathway in the T-cell to begin differentiation and to produce cytotoxic cytokines (2). Simultaneously, PD-1 receptors are activated by interactions between the antigen and the PD-1 and PD-2 ligands. The PD-1 ligands send inhibitory signals to various pathways of T-cells, such as signals inhibiting cytokine production (1). In healthy T-cells inhibition of these pathways counters the strong activation signaled by antigen-specific receptors. This prevents excessive cytokine production which could destroy healthy cells.
Analysis of T-cells in mice exposed to chronic and acute strains of lymphocytic choriomeningitis virus (LCMV) revealed a significant difference in expression of PD-1 receptors in exhausted T-cells. In mice exposed to the acute infection CD8+ T-cells show significantly less PD-1 receptor expression than T-cells in mice exposed to the chronic infection (1). These results suggest an up-regulation in transcription of pdcd1 gene which encodes for the PD-1 receptor.
In a separate study, mice chronically infected with LCMV were treated with PD-1 blocking peptides; this reduced the presence of the virus (5). The decreased viral presence indicates greater activation of CD8+ T-cells. With fewer PD-1 receptors less inhibition occurred allowing for the production of more cytotoxic cytokines. By decreasing inhibitory signals from PD-1 receptors the T-cells continued to target infected cells overcoming certain aspects of their exhausted state.
PD-1 expression is conserved in various chronic infections such as HIV, LCMV, Hepatitis C Virus and more (6). Regulation of PD-1 plays a key role in inhibiting T-cell activity during chronic infection. The question remains of how PD-1 expression increases during chronic infections.
Demethylation of pdcd1 locus In analyzing overexpression of PD-1 receptors in exhausted T-cells, researchers have turned to examining the pdcd1 locus. To compare methylation at the pdcd1 locus in acute and chronic infections, CD8+ T-cells from mice infected with acute and chronic LCMV were exposed to DNase (1). LCMV specific Thy1.1 marked CD8+ T-cells were introduced into wild-type mice. The mice were then infected with either an acute or chronic strain of LCMV. Four or nine days after infection the virus specific CD8+ T-cells were isolated through column purification with a biotinylated antibody specific to the Thy1.1 markers. A DNase I hypersensitivity assay was then performed on the pdcd1 locus of the isolated cells. Because DNase targets demethylated regions of DNA, the amount of digested DNA indicated the amount of demethylated DNA accessible to DNase. Results showed that the DNA in CD8+ T-cells from chronically infected mice were significantly more susceptible to DNase at the pdcd1 locus, suggesting more demethylation at the locus than during acute infection or in naïve T-cells analyzed before infection as a control. Demethylation of the pdcd1 locus and the consequential accessibility for transcriptional proteins, offers an explanation for the greater expression of PD-1 receptors during chronic infections like HIV.
To confirm the correlation of demethylation at the pdcd1 locus with the increased expression of PD-1 receptors, real time PCR was performed on PD-1 mRNA transcripts retrieved from a naïve T cells and from an antigen-specific memory T-cells (1). The data revealed that the naïve T-cells had significantly less PD-1 mRNA compared to effector T-cells. Bisulfite sequencing of the naïve and effector cells revealed more cysteine methylation in the pdcd1 locus of naïve cells compared to effector cells. The inverse relationship between methylation and the presence of PD-1 mRNA transcript suggests regulation of the transcription of the pdcd1 locus through methylation.
This study also tested PD-1 restimulation (1). CD8+ T-cells were removed from mice with either acute or chronic infection during a period of reduced PD-1 expression. The cells were then exposed to gp33, a peptide which binds to and activates LCMV antigen-specific receptors. Cytometric analysis was performed at 0, 6 and 12 hours to determine PD-1 expression over time. The CD8+ cells from chronic infections showed greater PD-1 expression than the cells from the acute infections. Since enough time passed between periods of stimulation to allow for new generations of T-cells to be produced, this evidence suggests that demethylation of pdcd1 is heritable through cell replication and maintained during differentiation in chronic infections.
This implies that CD8+ antigen-specific cells have altered methylation patterns which do not revert back to original patterns. So even though antigen-specific T-cells replicate and differentiate during chronic infection they express an exhausted phenotype due to the inherited methylation patterns which promote increased expression of inhibitory receptors.
Thus exhausted antigen-specific T-cells fail to respond to the presence of antigens and eliminate infected cells due to the premature dampening of effector response with the excessive inhibitory signals from PD-1 receptors (1) as depicted in Figure 1. Since PD-1 overexpression is conserved during exhaustion in many chronic infections similar demethylation of the pdcd1 locus may also be responsible for increased transcription and expression in these infections.
Figure 1 depicts some effects of pdcd1 methylation A) When pdcd1 is not methylated transcription factors can access the DNA and expression more PD-1 receptors. HIV specific CD8 co-receptors and T cell receptors respond to the HIV antigens produced by infected cell. In response these receptors signal the production of cytokines and the differentiation of the cell into an effector cell. Simultaneously PD-1 receptors inhibit the activation of cytokines. With demethylation of pdcd1 more PD-1 receptors inhibit cytokine production preventing the T-cell from eliminating the infected cell. B) When pdcd1 is methylated, the region is wrapped around histone to prevent the transcription factor from expressing the gene. As a result fewer PD-1 receptors are expressed and the T cell can successfully activate cytokine production.
Other transcriptional modifications Mounting evidence suggest that the transcriptional program of exhausted T-cells differs significantly from functional T-cells (6). Besides up-regulation of transcription at the pdcd1 locus, down-regulation of Dnmt3a2, an important protein for regulating methylation at CpG structures such as found in the pdcd1 locus, has been observed; methylation at CpG sites upstream of the transcriptional region for Dnmt3a2 is thought to cause the decreased expression by limiting the access of transcription factors to the DNA (1). The consequential decreased ability to regulate transcription through methylation may contribute to the inability of T-cells to produce cytokines or otherwise respond to antigens. Meanwhile, methylation and deacetylation has been observed to cause CD28 silencing (7) inhibiting Helper T-cells in recruiting CD8+ T-cells to the site of infection. Aside from the PD-1 pathway other areas of the genome experiencing methylation and deacetylation may be involved in further epigenetic transcriptional modification which contribute to functional limitations in exhausted T-cells.
Telomere length in CD8+ T cells
Telomere length Another characteristic of exhausted HIV-specific T-cells is shortened telomeres (7). Although the full purpose of telomeres is still largely uncertain, recent immunological studies imply that telomere shortening may contribute to the exhausted state of T-cells (8). This has been observed specifically in human patients with HIV (9). Telomere shortening has often been associated with the inability of a cell to divide, offering a potential explanation to the limited proliferation of exhausted CD8+ T-cells during chronic HIV infection (8).
To search for notable differences in size, one study compared the lengths of HIV specific CD8+ T-cells from a pool of HIV patients (10). The pool contained patients known as progressors, who experienced the continued immune system failures observed in advancing HIV infection and AIDS development. The pool also contained controllers, rare patients whose immune systems slowed the progression of the infection. By flow cytometry it was determined that CD8+ T-cells from progressors displayed a median absolute telomere length of 5.8 kb with a range 4.8-6.4 kb while HIV-1–specific CD8+ T-cells from controllers displayed a median absolute telomere length of 7.4 kb with a range of 6.3-12.9 kb (10). The significant difference in telomere length indicated the shorter telomeres found in progressors whose T-cell pool was composed of significantly higher proportion of exhausted T-cells.
The study further indicated that controllers experienced constitutive antigen-independent telomerase activity in comparison to significantly lower activity in progressors (10). It should also be noted that only moderately increased activity was observed in comparison to the longer lengths of telomeres, implying that telomerase is one of several mechanisms regulating the length of telomeres.
Effects of telomere length on T cell proliferation A key feature of exhaustion is the decreased ability of T-cells to proliferate and the subsequent weakening the immune response to antigens. In a seven person study of HIV specific CD8+ T-cells it was determined proliferating cells had longer telomere length than non-proliferating cells (10). This suggests that even during chronic infections longer telomeres stabilize the ability of cells to proliferate.
Another study confirmed this finding. A group of HIV specific CD8+ T cells were transduced with hTERT by viral injection into polyclonal human cell colonies (9). hTERT is a gene encoding for human telomerase and the transgene construct allowed for the transfected cells to express the telomerase constitutively. The colonies were allowed to grow in vivo with periodic restimulation of the receptors with antigen-like peptides. Duration of continued proliferation was measured for the colony and periodic samples were collected to measure telomere length by TRF assay. In comparison to a control colony with no hTERT injection, the consecutive transcription of telomerase allowed the transgene colony to continue proliferating for several hundred days longer than the control colony. Although telomeres in the transgene colony eventually shortened and the cells developed exhausted then senescent profiles, the experiment demonstrates that increased telomerase activity prolonged cell function. This indicates that exhaustion may be caused in part by reduced expression of telomerase.
This is reaffirmed by studies showing that telomeres do not shorten during acute infections due to an up-regulation of telomerase activity (11). It has also been noted that during prolonged infection T-cells are unable to continue up-regulating telomerase activity (11). Indeed telomere length in exhausted T-cells has be observed to approach the Hayflick limit, the minimal telomere length required to continue proliferation (12). However, if the lack of telomerase was the only cause of telomere shortening one would expect the addition of telomerase to allow the cells to continue dividing for a normal cell lifespan unlike described in the previous experiment.
Besides loss of telomerase function, cell exhaustion might be caused in part by loss of a group of proteins known as the shelterin complex. The shelterin complex interacts with the end of DNA strands to protect the sequence and regulate telomerase activity (12). Without the shelterin complex, the cell cannot effectively recruit telomerase and is unable to counter telomere shortening (12). The loss of shelterin from telomeres is specific to HIV infection and may be a unique contributor to exhaustion for this virus (12). As observed in many diseases and infections the shortening of telomeres leads to a decreased ability to proliferate.
PD-1 regulation of telomere length Although the direct mechanism has yet to be elucidated, PD-1 is suspected of having a regulatory role in telomerase activity. When HIV specific CD8+ T-cells were exposed to PD-1 blocking molecules, telomerase activity and telomere length increased (12). It should be noted that during this same study, blocking PD-1 signaling did not return normal shelterin complex expression. Shelterins may be regulated by another pathway which malfunctions during exhaustion or may not be replaceable after initial loss.
Figure 2 summarizes the discussed epigenetic pathways which contribute to cell exhaustion in CD8+ HIV-specific T-cells. It should be noted that the telomere related pathway has been observed thus far in HIV-specific T-cells only. The overexpression of PD-1 is conserved between many chronic infections including HIV.
Summary
Epigenetics offers mechanisms which may contribute to maintenance of exhaustion in T-cells during HIV infection. Current studies suggest that demethylation of the pdcd1 locus increases the expression of the PD-1 inhibitory receptors on CD8+ T-cells leading to excessive inhibitory signaling in effector cells and failure to eliminate infected cells as a consequence (Figure 2). The question remains on what causes the increased demethylation of the pdcd1 locus, for which no mechanisms have been confirmed. Because the overexpression of PD-1 in exhausted cells is conserved between types of chronic infections, demethylation may be conserved as well. Further questions remain concerning the degree to which methylation of the genome is altered due to observed transcriptional differences in other proteins essential for cytokinetic function. Such methylation may be caused by PD-1 signaling, methylation pathways or an external factor.
Meanwhile, telomere shortening and the decrease in telomerase activity is also related to HIV specific exhaustion (Figure 2). Telomere shortening is consistent with the slowed replication of T-cells in the presence of a chronic infection. However, the notable decrease in telomerase activity and the loss of the protective shelterin complex suggest other mechanisms may hasten T-cell exhaustion during infection. Further research should be conducted to determine how PD-1 signaling interferes with telomerase activity and what regulating forces control the expression of shelterin.
Overall, the mechanisms of exhaustion in HIV have yet to be completely understood at a molecular level. While epigenetics is strongly implicated in exhaustion the complete picture of viral components and other proteins involved remains elusive. Continued research into the epigenetic mechanisms and regulators offer promising therapies and vaccines to counter HIV.
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