- Email: [email protected]
CD38: Modulating Histone Methyltransferase EZH2 Activity in SLE
TREIMM 1655 No. of Pages 3
Trends in Immunology
Spotlight
CD38: Modulating Histone Methyltransferase EZH2 Activity in SLE Paramita Chakraborty1 and Shikhar Mehrotra1,* To keep autoreactive T cells under control in SLE patients, immunosuppressive regimens are used, which can increase susceptibility to bacterial and viral infections. Recently, Katsuyama et al., demonstrated that the CD38/NAD/ Sirtuin1/EZH2 axis reduces cytolytic CD8+ T cell function and might be targeted to overcome incidence of infections. Systemic lupus erythematosus (SLE) is an autoimmune disease where mostly macromolecules with proteins and nucleic acid constituents serve as auto-antigens and trigger the patients’ T and B cells to mount a self-destructive immune response. High-affinity antibodies to doublestranded DNA are characteristic hallmarks of human and murine SLE and can affect the skin, joints, kidneys, brain, and\or other organs [1]. While immunosuppressive drugs are used to blunt the effects of autoreactive immune cells, they can also result in increased susceptibility to bacterial or viral infections, representing a major cause for morbidity and mortality [2]. The mechanisms responsible for such increased susceptibility to infection in SLE patients are incompletely understood and efforts are underway to examine how suppressed T cells might be reprogrammed to reduce infections [3]. CD8+ cytotoxic T cells from SLE patients exhibit reduced effector function and cytolytic activity relative to those from healthy individuals [3]. A recent study by Katsuyama et al. found that cell surface
expression of CD38 was enhanced in CD4+, and CD8+ T cell subsets from SLE patients relative to healthy controls [4]. CD38 is not only a T cell activation molecule, but is also a noncanonical ectonucleotidase, that in conjunction with CD73, leads to the generation of adenosine [5]. Moreover, CD38 possesses nicotinamide adenine dinucleotide (NAD) glycohydrolase activity and the expression of CD38 has been inversely correlated to intrinsic concentrations of NAD+, a cofactor that serves as substrate for histone deacetylase (HDAC) SIRT1, in both murine and human cells [5].
execution of cytotoxic CD8+ T cell functions [4]. Furthermore, the authors assessed the expression of transcription factors T-BET, RUNX3, and EOMES (shown to control the expression of the above-mentioned effector molecules in murine models). While CD8+CD38hi T cells displayed low expression of T-BET, RUNX3, and EOMES proteins in healthy controls, only RUNX3 expression was decreased in CD8+CD38hi T cells from patients with SLE relative to healthy individuals [4].
Our group recently showed that CD38 expression induced on melanoma epitopereactive CD4+ T cells could dampen its ability to control established murine tumors by altering NAD+-dependent SIRT1 activity [6]. Furthermore, studies in different experimental disease models indicate that CD38 is a potent immunomodulator and a putative target for reprogramming the immune metabolic axis of T cells, altering their effector function [5–7]. The coexpression of CD38 and PD1 on peripheral CD8+ T cells or tumor infiltrating lymphocytes has also been correlated with tumor/node/ metastasis classification and clinical stage in pancreatic cancer patients [8]. Thus, strategies that can selectively limit CD38 expression on T cells may become important when attempting to overcome T cell (both CD8+ and CD4+) dysfunctionality and improve tumor control. However, its role in altering autoimmunity or infection has not been addressed.
Epigenetic mechanisms regulate gene expression either directly, by modifying DNA (DNA methylation), or indirectly, by modifying chromatin. Chromatin functionality and structure are tightly linked to covalent modification in histones. These modifications are generally classified as repressing or activating, generally correlating with gene silencing or induction, respectively. Among them, histone acetylation is associated with gene expression and is regulated by the action of histone acetyltransferases (HATs) and HDACs. The role of the NAD+/Sirt1 axis in regulating methylation has been examined; for instance, mixed-lineage leukemia (MLL1), a factor that is critically involved in oscillatory methylation of histone H3 at lysine K4 residue in circadian gene promoters can undergo NAD+-dependent deacetylation by SIRT1, leading to cyclic inhibition of its methyltransferase activity in various cell types [9]. Thus, a crosstalk between acetylation and methylation, that in turn links cellular metabolic state to epigenetic control, has been established.
The present study by Katsuyama et al. identified a population of SLE patients exhibiting increased risk of infections and expressing high amounts of CD38 on CD8+ cytotoxic T cells (Figure 1). CD8+CD38hi T cells also displayed reduced expression of granzyme A (GZMA), granzyme B (GZMB), perforin (PRF1), and interferon-gamma (IFN-γ) molecules relative to healthy individuals and these factors are needed for the
Given the inverse correlation between CD38 and NAD+, the authors further interrogated if SIRT1 mediated deacetylation of Enhancer of Zeste Homolog 2 (EZH2), which epigenetically modulates the expression of several molecules (e.g., T-bet, RUNX3, and EOMES), by trimethylating H3K27me3, and whether it played a role in the functional outcome (i.e., repressed effector function) of CD8+CD38hi T cells. Trends in Immunology, Month 2020, Vol. xx, No. xx
1
Trends in Immunology
SLE patients, chromatin accessibility did not parallel gene expression data observed in SLE patients. This might be likely due to the basal activated state of autoreactive T cells in SLE patients when compared with normal healthy individuals, but further experiments are warranted.
Trends in Immunology
Figure 1. A Correlation between CD38 Expression and EZH2 Activity in CD8+ T Cells from SLE Patients. Schematic representation of the CD38/NAD/Sirtuin1/EZH2 axis that can increase methylation of effector genes and, in turn, reduce cytolytic CD8 + T cell functions. Strategies to inhibit EZH2 to restore cytolytic function might offer promise to overcome incidence of infections in immunosuppressed SLE patients [4]. Abbreviations: Ac, acetylation; EZH2, Enhancer of Zeste Homolog 2; Me, methylation; NAD, nicotinamide adenine dinucleotide; SLE, systemic lupus erythematosus. This figure was created using BioRender (https://biorender.com/).
Overall, this study by the Tsokos group, comprehensively identified an underlying mechanism responsible for decreased cytotoxicity of CD8+CD38hi T cells from SLE patients [4]. Presumably, this mechanism might be pharmacologically targeted in the future to overcome susceptibility to infection. However, inhibiting EZH2 can not only increase effector and cytotoxic T cell functions, but can also impair suppressive functions of tolerance-inducing regulatory T cells, which can lead to spontaneous autoimmunity [11]. Thus, a balance between autoimmunity and overcoming infections must be ensured and a cautious regimen needs to be ascertained in relevant murine models to assess differences in infection control when aiming to target EZH2. Regardless, this study opens exciting avenues that could be further explored when investigating clinical paths. Acknowledgments
+
hi
Indeed, CD8 CD38 T cells from both SLE patients (n = 5) and normal healthy donors (n = 7) showed increased expression of EZH2 compared with CD8+CD38lo T cells [4]. Their data also indicated that inhibition of SIRT1 deacetylase activity in CD38lo (which are NADhi) increased methyltransferase activity of EZH2. Conversely, CD38hi (and NADlo) T cells exhibited more EZH2 acetylation and stable methyltransferase activity on its targets; this was evidenced by the increased amounts of H3K27me3 on CD8+CD38hi as compared with CD8+CD38lo T cells. Furthermore, treatment of CD38hi T cells with the EZH2 inhibitor GSK126 inhibited methylation and restored their effector function (IFN-γ, 2
Trends in Immunology, Month 2020, Vol. xx, No. xx
GZM) and cytolytic activity (CD107a) in vitro [4]. Since differences in NAD+ can alter chromatin organization [10], the authors examined whether differences in CD38 expression restricted chromatin accessibility to RUNX3, EOMES, and TBX21 loci, by performing assay for transposase accessible chromatin using sequencing (ATAC-Seq) to assess genome-wide chromatin accessibility changes in the T cells. While correlation heatmaps and principal component analysis showed variation in chromatin accessibility between CD8 + CD38 lo and CD8 + CD38 hi T cell populations between healthy subjects and
Authors acknowledge support from National Institutes of Health (NIH) R01 CA236379, NIH PO1 CA203628, Hollings Cancer Center (supported in part by National Institutes of Health Grant P30 CA138313), and South Carolina Center of Biomedical Research Excellence (COBRE) in Oxidants, Redox Balance, and Stress Signaling, and NIH Grant 5P20GM103542. 1
Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA *Correspondence: [email protected] (S. Mehrotra). https://doi.org/10.1016/j.it.2020.01.008 © 2020 Elsevier Ltd. All rights reserved.
References 1. Fors Nieves, C.E. and Izmirly, P.M. (2016) Mortality in systemic lupus erythematosus: an updated review. Curr. Rheumatol. Rep. 18, 21
Trends in Immunology
2.
3.
4.
Danza, A. and Ruiz-Irastorza, G. (2013) Infection risk in systemic lupus erythematosus patients: susceptibility factors and preventive strategies. Lupus 22, 1286–1294 Kis-Toth, K. et al. (2016) Selective loss of signaling lymphocytic activation molecule family member 4-positive CD8+ T cells contributes to the decreased cytotoxic cell activity in systemic lupus erythematosus. Arthritis Rheumatol. 68, 164–173 Katsuyama, E. et al. (2020) The CD38/NAD/SIRTUIN1/ EZH2 axis mitigates cytotoxic CD8 T cell function and
5.
6.
7.
identifies patients with SLE prone to infections. Cell Rep. 30, 112–123 Hogan, K.A. et al. (2019) The multi-faceted ecto-enzyme CD38: roles in immunomodulation, cancer, aging, and metabolic diseases. Front. Immunol. 10, 1187 Chatterjee, S. et al. (2018) CD38-NAD(+)axis regulates immunotherapeutic anti-tumor T cell response. Cell Metab. 27, 85–100 Verma, V. et al. (2019) PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nat. Immunol. 20, 1231–1243
8.
Zhang, M. et al. (2019) Prognostic values of CD38(+) CD101(+)PD1(+)CD8(+) T cells in pancreatic cancer. Immunol. Investig. 48, 466–479 9. Sassone-Corsi, P. (2012) Minireview: NAD+, a circadian metabolite with an epigenetic twist. Endocrinology 153, 1–5 10. Kim, M.Y. et al. (2004) NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell 119, 803–814 11. DuPage, M. et al. (2015) The chromatin-modifying enzyme Ezh2 is critical for the maintenance of regulatory T cell identity after activation. Immunity 42, 227–238
Trends in Immunology, Month 2020, Vol. xx, No. xx
3
Trends in Immunology
Spotlight
CD38: Modulating Histone Methyltransferase EZH2 Activity in SLE Paramita Chakraborty1 and Shikhar Mehrotra1,* To keep autoreactive T cells under control in SLE patients, immunosuppressive regimens are used, which can increase susceptibility to bacterial and viral infections. Recently, Katsuyama et al., demonstrated that the CD38/NAD/ Sirtuin1/EZH2 axis reduces cytolytic CD8+ T cell function and might be targeted to overcome incidence of infections. Systemic lupus erythematosus (SLE) is an autoimmune disease where mostly macromolecules with proteins and nucleic acid constituents serve as auto-antigens and trigger the patients’ T and B cells to mount a self-destructive immune response. High-affinity antibodies to doublestranded DNA are characteristic hallmarks of human and murine SLE and can affect the skin, joints, kidneys, brain, and\or other organs [1]. While immunosuppressive drugs are used to blunt the effects of autoreactive immune cells, they can also result in increased susceptibility to bacterial or viral infections, representing a major cause for morbidity and mortality [2]. The mechanisms responsible for such increased susceptibility to infection in SLE patients are incompletely understood and efforts are underway to examine how suppressed T cells might be reprogrammed to reduce infections [3]. CD8+ cytotoxic T cells from SLE patients exhibit reduced effector function and cytolytic activity relative to those from healthy individuals [3]. A recent study by Katsuyama et al. found that cell surface
expression of CD38 was enhanced in CD4+, and CD8+ T cell subsets from SLE patients relative to healthy controls [4]. CD38 is not only a T cell activation molecule, but is also a noncanonical ectonucleotidase, that in conjunction with CD73, leads to the generation of adenosine [5]. Moreover, CD38 possesses nicotinamide adenine dinucleotide (NAD) glycohydrolase activity and the expression of CD38 has been inversely correlated to intrinsic concentrations of NAD+, a cofactor that serves as substrate for histone deacetylase (HDAC) SIRT1, in both murine and human cells [5].
execution of cytotoxic CD8+ T cell functions [4]. Furthermore, the authors assessed the expression of transcription factors T-BET, RUNX3, and EOMES (shown to control the expression of the above-mentioned effector molecules in murine models). While CD8+CD38hi T cells displayed low expression of T-BET, RUNX3, and EOMES proteins in healthy controls, only RUNX3 expression was decreased in CD8+CD38hi T cells from patients with SLE relative to healthy individuals [4].
Our group recently showed that CD38 expression induced on melanoma epitopereactive CD4+ T cells could dampen its ability to control established murine tumors by altering NAD+-dependent SIRT1 activity [6]. Furthermore, studies in different experimental disease models indicate that CD38 is a potent immunomodulator and a putative target for reprogramming the immune metabolic axis of T cells, altering their effector function [5–7]. The coexpression of CD38 and PD1 on peripheral CD8+ T cells or tumor infiltrating lymphocytes has also been correlated with tumor/node/ metastasis classification and clinical stage in pancreatic cancer patients [8]. Thus, strategies that can selectively limit CD38 expression on T cells may become important when attempting to overcome T cell (both CD8+ and CD4+) dysfunctionality and improve tumor control. However, its role in altering autoimmunity or infection has not been addressed.
Epigenetic mechanisms regulate gene expression either directly, by modifying DNA (DNA methylation), or indirectly, by modifying chromatin. Chromatin functionality and structure are tightly linked to covalent modification in histones. These modifications are generally classified as repressing or activating, generally correlating with gene silencing or induction, respectively. Among them, histone acetylation is associated with gene expression and is regulated by the action of histone acetyltransferases (HATs) and HDACs. The role of the NAD+/Sirt1 axis in regulating methylation has been examined; for instance, mixed-lineage leukemia (MLL1), a factor that is critically involved in oscillatory methylation of histone H3 at lysine K4 residue in circadian gene promoters can undergo NAD+-dependent deacetylation by SIRT1, leading to cyclic inhibition of its methyltransferase activity in various cell types [9]. Thus, a crosstalk between acetylation and methylation, that in turn links cellular metabolic state to epigenetic control, has been established.
The present study by Katsuyama et al. identified a population of SLE patients exhibiting increased risk of infections and expressing high amounts of CD38 on CD8+ cytotoxic T cells (Figure 1). CD8+CD38hi T cells also displayed reduced expression of granzyme A (GZMA), granzyme B (GZMB), perforin (PRF1), and interferon-gamma (IFN-γ) molecules relative to healthy individuals and these factors are needed for the
Given the inverse correlation between CD38 and NAD+, the authors further interrogated if SIRT1 mediated deacetylation of Enhancer of Zeste Homolog 2 (EZH2), which epigenetically modulates the expression of several molecules (e.g., T-bet, RUNX3, and EOMES), by trimethylating H3K27me3, and whether it played a role in the functional outcome (i.e., repressed effector function) of CD8+CD38hi T cells. Trends in Immunology, Month 2020, Vol. xx, No. xx
1
Trends in Immunology
SLE patients, chromatin accessibility did not parallel gene expression data observed in SLE patients. This might be likely due to the basal activated state of autoreactive T cells in SLE patients when compared with normal healthy individuals, but further experiments are warranted.
Trends in Immunology
Figure 1. A Correlation between CD38 Expression and EZH2 Activity in CD8+ T Cells from SLE Patients. Schematic representation of the CD38/NAD/Sirtuin1/EZH2 axis that can increase methylation of effector genes and, in turn, reduce cytolytic CD8 + T cell functions. Strategies to inhibit EZH2 to restore cytolytic function might offer promise to overcome incidence of infections in immunosuppressed SLE patients [4]. Abbreviations: Ac, acetylation; EZH2, Enhancer of Zeste Homolog 2; Me, methylation; NAD, nicotinamide adenine dinucleotide; SLE, systemic lupus erythematosus. This figure was created using BioRender (https://biorender.com/).
Overall, this study by the Tsokos group, comprehensively identified an underlying mechanism responsible for decreased cytotoxicity of CD8+CD38hi T cells from SLE patients [4]. Presumably, this mechanism might be pharmacologically targeted in the future to overcome susceptibility to infection. However, inhibiting EZH2 can not only increase effector and cytotoxic T cell functions, but can also impair suppressive functions of tolerance-inducing regulatory T cells, which can lead to spontaneous autoimmunity [11]. Thus, a balance between autoimmunity and overcoming infections must be ensured and a cautious regimen needs to be ascertained in relevant murine models to assess differences in infection control when aiming to target EZH2. Regardless, this study opens exciting avenues that could be further explored when investigating clinical paths. Acknowledgments
+
hi
Indeed, CD8 CD38 T cells from both SLE patients (n = 5) and normal healthy donors (n = 7) showed increased expression of EZH2 compared with CD8+CD38lo T cells [4]. Their data also indicated that inhibition of SIRT1 deacetylase activity in CD38lo (which are NADhi) increased methyltransferase activity of EZH2. Conversely, CD38hi (and NADlo) T cells exhibited more EZH2 acetylation and stable methyltransferase activity on its targets; this was evidenced by the increased amounts of H3K27me3 on CD8+CD38hi as compared with CD8+CD38lo T cells. Furthermore, treatment of CD38hi T cells with the EZH2 inhibitor GSK126 inhibited methylation and restored their effector function (IFN-γ, 2
Trends in Immunology, Month 2020, Vol. xx, No. xx
GZM) and cytolytic activity (CD107a) in vitro [4]. Since differences in NAD+ can alter chromatin organization [10], the authors examined whether differences in CD38 expression restricted chromatin accessibility to RUNX3, EOMES, and TBX21 loci, by performing assay for transposase accessible chromatin using sequencing (ATAC-Seq) to assess genome-wide chromatin accessibility changes in the T cells. While correlation heatmaps and principal component analysis showed variation in chromatin accessibility between CD8 + CD38 lo and CD8 + CD38 hi T cell populations between healthy subjects and
Authors acknowledge support from National Institutes of Health (NIH) R01 CA236379, NIH PO1 CA203628, Hollings Cancer Center (supported in part by National Institutes of Health Grant P30 CA138313), and South Carolina Center of Biomedical Research Excellence (COBRE) in Oxidants, Redox Balance, and Stress Signaling, and NIH Grant 5P20GM103542. 1
Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA *Correspondence: [email protected] (S. Mehrotra). https://doi.org/10.1016/j.it.2020.01.008 © 2020 Elsevier Ltd. All rights reserved.
References 1. Fors Nieves, C.E. and Izmirly, P.M. (2016) Mortality in systemic lupus erythematosus: an updated review. Curr. Rheumatol. Rep. 18, 21
Trends in Immunology
2.
3.
4.
Danza, A. and Ruiz-Irastorza, G. (2013) Infection risk in systemic lupus erythematosus patients: susceptibility factors and preventive strategies. Lupus 22, 1286–1294 Kis-Toth, K. et al. (2016) Selective loss of signaling lymphocytic activation molecule family member 4-positive CD8+ T cells contributes to the decreased cytotoxic cell activity in systemic lupus erythematosus. Arthritis Rheumatol. 68, 164–173 Katsuyama, E. et al. (2020) The CD38/NAD/SIRTUIN1/ EZH2 axis mitigates cytotoxic CD8 T cell function and
5.
6.
7.
identifies patients with SLE prone to infections. Cell Rep. 30, 112–123 Hogan, K.A. et al. (2019) The multi-faceted ecto-enzyme CD38: roles in immunomodulation, cancer, aging, and metabolic diseases. Front. Immunol. 10, 1187 Chatterjee, S. et al. (2018) CD38-NAD(+)axis regulates immunotherapeutic anti-tumor T cell response. Cell Metab. 27, 85–100 Verma, V. et al. (2019) PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nat. Immunol. 20, 1231–1243
8.
Zhang, M. et al. (2019) Prognostic values of CD38(+) CD101(+)PD1(+)CD8(+) T cells in pancreatic cancer. Immunol. Investig. 48, 466–479 9. Sassone-Corsi, P. (2012) Minireview: NAD+, a circadian metabolite with an epigenetic twist. Endocrinology 153, 1–5 10. Kim, M.Y. et al. (2004) NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell 119, 803–814 11. DuPage, M. et al. (2015) The chromatin-modifying enzyme Ezh2 is critical for the maintenance of regulatory T cell identity after activation. Immunity 42, 227–238
Trends in Immunology, Month 2020, Vol. xx, No. xx
3