Glutaminase 2 Induces Interleukin-2 Production in CD4+ T Cells by Supporting Antioxidant Defense

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Citation Orite, Seo Yeon Kim. 2019. Glutaminase 2 Induces Interleukin-2 Production in CD4+ T Cells by Supporting Antioxidant Defense. Master's thesis, Harvard Medical School.

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Glutaminase 2 Induces Interleukin-2 Production in CD4+ T cells by

Supporting Antioxidant Defense

Seo Yeon Kim Orite

A Thesis Submitted to the Faculty of

The Harvard Medical School

in Partial Fulfillment of the Requirements

for the Degree of Master of Medical Sciences in Immunology

Harvard University

Boston, Massachusetts.

May, 2019

Thesis Advisor: Dr. Tsokos Seo Yeon Kim Orite

Glutaminase 2 Induces Interleukin-2 Production in CD4+ T cells by Supporting Antioxidant Defense

Abstract

Metabolic abnormalities of T cells contribute to pathogenesis of many autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Rapidly proliferating effector T cells often utilize glutamine as an energy source to meet high energy demands. Glutaminase (Gls) is the first enzyme in the glutaminolysis pathway converting glutamine to glutamate. In addition to providing energy through glutamine processing, Gls also supports antioxidant defense against cellular stress by driving glutathione (GSH) synthesis. There are two isoforms of Gls that are differentially regulated. Gls2, but not Gls1, induced interleukin 2 (IL-2) production in CD4+ T cells of mouse and human and Jurkat cell line. Upregulation of Gls2 expression increased IL-2 production at the protein and the transcriptional levels. Moreover, Gls2 overexpression in CD4+

T cells lowered cellular oxidative stress. Taken together, I propose that Gls2 induces IL-2 production by reducing intracellular reactive oxygen species (ROS) level known to suppress IL-2 production. In addition, p53 plays an important role in this Gls2-mediated IL-2 production pathway by directly targeting the Gls2 gene. Finally, patients with SLE express reduced levels of p53 and GLS2 protein expression compared to healthy controls. These findings suggest that Gls2- mediated IL-2 induction pathway is disrupted in patients with SLE. However, further investigation is required to determine the exact molecular mechanism of this pathway and to evaluate therapeutic potential of exogenous Gls2 delivery.

Table of Contents

Chapter 1: Background ···················································································· 1 1.1: Aberrant T Cell Function in Systemic Lupus Erythematosus (SLE) ···················· 1 1.2: T Cell Metabolism Dysfunction in SLE ························································ 2 1.3: Function of Interleukine-2 (IL-2) ································································ 4 1.4: Reduced IL-2 Production in SLE ································································ 4 1.5: GSH, ROS, and IL-2 ··············································································· 5 1.6: Glutaminolysis ······················································································· 6 1.7: p53 and GLS2 ························································································ 8 1.8: Research Question ·················································································· 9 Chapter 2: Materials and Methods ···································································· 10 Chapter 3: Results and Discussion ····································································· 15 3.1: Results ······························································································· 15 3.2: Discussion ··························································································· 26 Chapter 4: Limitations and Perspectives ····························································· 31 Chapter 5: Bibliography ················································································· 35 Appendix: Demographic Information for Human Subjects ······································ 39

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List of Figures

Figure 1.1: Molecular Targets of Oxidative Stress in T cells. Oxidative stress, ROS, originate primarily in the mitochondria. Oxidative stress and associated changes in calcium storage also activate CREM, which suppresses IL-2 and enhances IL-17 promoter activity. The figure has been adopted and modified from Perl, 20131. This work is a derivative of Servier Medical Art. Servier

Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.

Figure 1.2: A General Scheme Summarizing Interaction of Glutaminolysis and Antioxidant

Defense in T cells. Glutamine processing supports antioxidant defense by promoting glutathione synthesis.

Figure 3.1: GLS2 Expression is Diminished in Patients with SLE. (A) Purified CD4+ T cells isolated from patients with SLE and matched healthy donors were stimulated in plate-bound anti- human CD3 (OKT3) and soluble anti-human CD28 overnight. Healthy donors are age-, sex- and race-matched to patients with SLE. GLS2 and beta actin expression were assessed by Western blotting. Representative data are shown. (B) Densitometry analysis from eight experiments are shown (*P<0.05, mean±s.e.m., two-tailed t-test, n=8).

Figure 3.2: Gls2 expression is diminished in CD4+ T cells isolated from MRL/MpJ-Faslpr

(MRL/lpr), a mouse model for systemic autoimmunity, compared to its control strain,

MRL/MpJ. (A) Purified CD4+ T cells isolated from 15 week-old MRL/MpJ and MRL/lpr mice were assessed by Western blot for Gls2 and beta actin expression. Representative data are shown.

(B) Densitometry analysis from two experiments are shown (mean±s.e.m., two-tailed t-test, n=2).

Figure 3.3: Gls2 Overexpression in Primary Mouse CD4+ T cell Induces IL-2 Production at both the Transcriptional and Protein Levels. (A) Gating strategy to isolate vector transfected, live, single cells for FACS analysis. DsRED positive cells were sorted prior to flow cytometry analysis. (B) Empty vector (Empty) or Gls2-expressing (Gls2) plasmid were transfected to naïve

CD4+ T cells from C57BL/6 mice that had been incubated under Th0 nonpolarizing conditions for 1.5 days. DsRED positive cells were sorted 24 hours post-transfection. The percentage of IL-

2-producing cells in the sorted cells were measured by flow cytometry. (C) Cumulative results of flow cytometry analysis on IL-2 producing CD4+ T cells (**P<0.01, mean±s.e.m., two-tailed t- test, n=6). (D) Cumulative results of IL-2 reporter activity measured by dual-luciferase assay

(**P<0.01, mean±s.e.m., two-tailed t-test, n=6).

Figure 3.4: GLS2 Overexpression Increases IL-2 Transcription in Jurkat Cells and Primary

Human CD4+ T Cells. (A) IL-2 promoter activity of Jurkat cell transfected with empty vector

(empty) and GLS2-expressing plasmid (GLS2 OE) are shown. After transfection, Jurkat cells were stimulated with soluble anti-human CD3 and anti-human CD28 for 2 days prior to analysis. Each dot represents an independent experiment. (B) IL-2 promoter activity of primary human CD4+ cell isolated from peripheral blood transfected with empty vector (empty) or GLS2-expressing plasmid

(GLS2 OE) are shown. After transfection, human CD4+ T cells were stimulated with plate-bound anti-human CD3 and soluble anti-human CD28 antibodies for 2 days prior to analysis (*P<0.05, mean±s.e.m., two-tailed t-test, n=3)..

Figure 3.5: GLS2 Overexpression Reduces Oxidative Stress in Human CD4+ T Cells. (A)

Gating strategy to isolate transfected, live, single human CD4+ T cells for flow cytometry analysis.

(B) Representative histogram of ROS in human CD4+ T cells transfected with empty vector

(Empty) or GLS2-expressing plasmid (GLS2). Non-transfected human CD4+ T cells were used as

v a control. (C) The percentage of ROS producing human CD4+ T cells were quantified by flow cytometry.

Figure 3.6: p53 Expression in Patients with SLE is Lower than Healthy Controls. (A) Gating strategy to isolate single, live CD4+ T cells (ZA-, CD3+, CD4+, CD8-) from PBMC. (B) The percentage of p53-producing cells among CD4+ T cells gated from PBMC of patients with SLE and matched healthy donors were measured by flow cytometry. Healthy donors are age-, sex- and race-matched to patients with SLE (*P<0.05, mean±s.e.m., Mann-Whitney test, n: HC=7;

SLE=11). (C) The MFI for p53 expression of human CD4+ T cells were quantified. Performed with help from Dr. Catalina Burbano.

Acknowledgements

I would like to thank everyone in Tsokos lab for teaching me various experimental protocols. I felt very comfortable sharing ideas and opinions about our research topics with them.

I am profoundly grateful to my mentor Dr. Nobuya Yoshida who has worked with me every day to produce this thesis. I would also like to thank Dr. Masataka Umeda and Dr. Catalina Burbano for their support and encouragement. Last but not least, I would like to give heartfelt thanks to my thesis advisor Dr. George Tsokos for giving me the opportunity to conduct this project in his lab.

Through weekly meetings, Dr. Tsokos gave me invaluable advices and guidance with my research project.

I should thank Dr. Shive Pillai and Dr. Michael Carroll, the directors the Immunology program at Harvard Medical School. With thoughtful feedbacks, Dr. Pillai and Dr. Carroll guided me throughout this program. I would also like to thank our program manager Selina Sarmiento for being approachable and providing clear instructions about everything related to this program.

This work was conducted with support from Students in the Master of Medical Sciences in

Immunology program of Harvard Medical School. The content is solely the responsibility of the author(s) and does not necessarily represent the official views of Harvard University and its affiliated academic health care centers.

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Chapter 1: Background

1.1: Aberrant T Cell Function in Systemic Lupus Erythematosus (SLE)

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that affects multiple organs. Genetic, environmental, immuno-regulatory, hormonal and epigenetic risk factors contribute to the onset and progression of SLE2. Although molecular presentations vary among patients affected by SLE, most of the patients develop autoantibodies immune complexes, autoreactive T cell and inflammatory cytokines2. Through several different mechanisms, these abnormalities cause inflammation of and damage to practically every organ. Due to its complex clinical manifestations, developing a targeted therapy for SLE has been challenging. Current treatment focuses on alleviating clinical symptoms through general immunosuppression3,4.

Unfortunately, only small portion of patients with SLE responded to most biologics. Belimumab is the only FDA approved targeted therapy for SLE treatment and it targets the B Lymphocyte stimulator (BLyS)5.

Although SLE was first recognized as a B cell mediated disease, more and more evidence suggests that T cells play a crucial role in pathogenesis of SLE6,7. SLE T cells display distinct abnormalities, which can be potential therapeutic targets. In homeostasis, activated T cells proliferate and differentiate into effector and memory T cells in order to fight against pathogens.

Effector T cells recruit other immune cells to clear infections efficiently8. Memory T cells allow immune system to recognize pathogens more readily in the future8. These T cell responses diminish once infected cells and foreign antigen are eliminated through apoptosis8. Moreover, the regulatory T cells prevent inappropriate immune responses and sustain self-tolerance8. However, in SLE, many of these self-tolerance mechanisms are disrupted, contributing to the pathogenesis of the disease. For example, SLE T cell differentiation is skewed to Th1 and Th17 effector subsets9.

In addition, regulatory T cell (Treg) numbers in patients with active SLE are lower than healthy control10. T cells isolated from patients with SLE are also known to provide excess help to B cells9.

Cytokine production also deviates in SLE T cells. The regulatory cytokine interleukin 2 (IL-2) production is reported to be reduced and pro-inflammatory interleukin 17 (IL-17) production is increased9.

1.2: T Cell Metabolism Dysfunction in SLE

Aberrant T cell metabolism accounts for the SLE T cell phenotype mentioned above11.

Targeting these abnormalities present new therapeutic approaches. Metabolic abnormalities in

SLE were first characterized in T cells in 200212. According to Gergely et al., T cells isolated from patients with SLE had hyperpolarized mitochondria characterized by decreased adenosine triphosphate (ATP) production and increased reactive oxygen species (ROS)12,13. Intracellular

ATP depletion also contributes to pathogenesis of SLE. Since ATP is required for apoptosis14,

ATP depletion seen in SLE T cells made SLE T cells more prone to necrosis.

Apoptosis is a nonlytic form of programmed cell death. Apoptosis is often referred as

“clean cell death” because it does not elicit inflammatory responses15. In contrast, necrosis is a lytic form of programmed cell death16. By releasing cellular contents including pro-inflammatory signals, necrosis leads to accumulation of inflammation seen in SLE12. In addition to promoting inflammation, increased necrosis causes overproduction of ROS. Necrosis can also result from high ROS level17. Another hallmark of SLE T cell metabolic abnormality is increased oxidative stress12. Both the total glutathione (GSH) level and ratio of glutathione (GSH) to glutathione disulphide (GSSG) in SLE T cells were reduced compared to healthy controls12. GSH is a powerful intracellular antioxidant that can clear ROS. Reduced GSH level in patients with SLE shows that

SLE T cells are incapable of neutralizing oxidative stress efficiently. One way that ROS contribute

2 to SLE phenotype is causing direct damage to DNA, generating neoantigens18. This ultimately results in autoantibody generation18. High oxidative stress can also contribute to SLE by modulating cytokine transcription. Oxidative stress increases calcium influx19,20 and activates cyclic AMP response-element modulator α (CREM"). This activation of CREM" reduces IL-2 production but enhances IL-17 production21,22 (Figure 1.1).

Oxidative Stress, ROS

Mitochondrion Increased Ca2+ storage Activation

CREM!

IL-2 IL-17

Nuclues

1.3: Function of Interleukine-2 (IL-2)

Since its discovery, IL-2 has been studied extensively. IL-2 plays many important roles in the regulation of the immune response. Released mostly by activated conventional T cells, IL-2 acts as a proliferation signal to CD4+ and CD8+ T cells during the immune response23. IL-2 supports survival of antigen-activated T cells by promoting anti-apoptotic protein Bcl-28.

IL-2 also has a regulatory function, and dysfunction of this role can be observed in many autoimmune diseases24–27. IL-2 downregulates immune response by eliminating activated T cells23.

IL-2 accomplishes this by upregulating FasL expression on activated T cells and initiates their apoptosis28,29. In mice, IL-2 also induces apoptosis of T cells by reducing expression of apoptotic inhibitors such as fas-associated death domain-like IL-1β–converting enzyme-inhibitor protein

(FLIP)28. In addition, IL-2 is required for proliferation and function of regulatory T cells23. Due to its critical role in immune tolerance and regulation, deletion of IL-2 or IL-2 receptor is known to cause autoimmune disease in mice and humans30,31. In fact, IL-2 deletion resulted in reduction of thymic Treg cell numbers in mice by half 32. Taken together, maintaining IL-2 levels is essential key to normal immune response and tolerance.

1.4: Reduced IL-2 Production in SLE

In the early 1980s scientists established that IL-2 levels were low in both lupus-prone mice and patients with SLE33–35. Limited amount of IL-2 in patients with SLE has many adverse effects.

With low IL-2, cytotoxic T cells are dysfunctional, increasing the chances of infection in patients36.

Low IL-2 also increases longevity of T cells including the autoreactive ones36. Further studies on

IL-2 production in SLE revealed that abnormal engagement of TCR and CD3 complex in SLE T cells increases Ca2+ influx and activity of Ca2+/calmodulin–dependent kinase IV (CaMK4)21,37.

CaMK4 then induces cyclic AMP response-element modulator α (CREM"), which suppresses IL-

2 transcription by directly binding to IL-2 promoter21.

There have been many attempts to deliver exogenous IL-2 to treat patients with SLE.

Recently, low dosage IL-2 treatment showed therapeutic effects on patients with SLE3. In a low- dose IL-2 clinical trial involving 5 patients, 1.5 million IU of human recombinant IL-2 was injected to patients subcutaneously for five days38. After five days of low dose IL-2 treatment, clinical symptoms improved and the Treg population was restored in patients with SLE without causing any noticeable side effects38. Another non-controlled clinical trial involving 38 patients showed even more promising results. In this clinical trial, 1 million IU of human recombinant IL-2 was injected subcutaneously every other day for two weeks39. Patients then took a two-week break from IL-2 administration before repeating two more cycles of injections39. After 12 weeks, the

SLE Disease Activity Index (SLEDAI) of the patients dropped by 4 points and the Treg population expanded39. Therefore, reconstituting IL-2 levels in patients with SLE has many therapeutic benefits and should be investigated more.

1.5: GSH, ROS, and IL-2

As briefly mentioned above, GSH is a crucial intracellular antioxidant. In the absence of oxidative stress, GSH stays in a reduced state40. GSH reduces ROS such as hydrogen peroxide and lipid peroxides with the assistance of GSH peroxidase40. As a result of oxidative stress, GSH becomes oxidized to GSSG. GSSG reductase then uses a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as the source of electrons to return GSSG to its reduced form,

GSH.40

Several lines of evidence support that managing oxidative stress can modulate T cell function and cytokine production. For example, in murine T cells, pharmaceutical inhibition of

GSH with L-Buthionine-S,R-sulfoximine (BSO) significantly reduced IL-2 production, while treatment with thiol N-acetylcysteine (NAC), a non-enzymatic antioxidant and precursor of GSH, restored IL-2 production to baseline41. NAC intake was able to correct metabolic abnormalities caused by high oxidative stress without any major side effect, even in patients with SLE42. With daily supplement of 2.4g of NAC, patients with SLE reduced disease severity within three months and FoxP3 expression in the Treg population was increased42. FoxP3 expression is a good indicator for normal Treg function because FoxP3 expression strengthens immuno-suppressive functions43.

Although the clinical trial data did not directly support the induction of IL-2 through ROS reduction, recovered Treg function may represent the result of restored IL-2 production.

1.6: Glutaminolysis

Glutaminolysis is a metabolic pathway that uses glutamine as an energy source.

Glutaminase is an enzyme that is involved in the first step of glutamine processing. There are two isoforms of glutaminase: glutaminase 1 (Gls1) and glutaminase 2 (Gls2). Both Gls1 and Gls2 catabolize glutamine into glutamate, but these enzymes are different in many ways. Gls1 and Gls2 are located in different chromosomes. According to NCBI, Gls1 is on chromosome 2 in humans and chromosome 1 in mice. Gls2 is located on chromosome 12 in humans and chromosome 10 in mice. In fact, a review by Mates et al emphasizes that GLS1 and GLS2 have an opposite effect on cancer progression. In tumor cells with dysregulated oncogene Myc, GLS1 expression was increased, whereas GLS2 was suppressed44. In a carcinoma cell line, GLS1 promoted progression of cancer cells while GLS2 repressed tumor growth45,46. In glioblastoma cells, selectively silencing

GLS1or overexpressing GLS2 reduce tumor cell proliferation47.

Glutaminolysis is largely explored in the context of cancer, but not much in autoimmune disease. Recently, Tsokos Lab found that glutaminase plays an important role in the pathogenesis of autoimmune disease. Kono et al. discovered that Gls1 is important for Th17 differentiation.

Inducible cAMP early repressor (ICER), which promotes Th17 cell differentiation48, enhances transcriptionally the expression of Gls149. Gls1 inhibition reduced Th17 differentiation in vitro and delayed disease progression in Experimental Autoimmune Encephalomyelitis (EAE)49. Taken together, this study demonstrated that glutaminolysis is a suitable target to treat autoimmune disease.

Glutamine processing through Gls2 may also play an important role in protection against autoimmunity via strengthening defense against oxidative stress (Figure 1.2). Glutamate, a molecule synthesized in the glutaminolysis pathway, serves as a precursor to GSH synthesis

Glutamine Glutamine Transporter

Glutamine Cysteine + Glycine Glutaminase Glutamate Glutathione (GSH) α-ketoglutarate

TCA cycle GSH ROS

GSSG None-Radicals

Figure 1.2: A General Scheme Summarizing Interaction of Glutaminolysis and Antioxidant Defense in T cells. Glutamine processing supports antioxidant defense via promoting glutathione synthesis.

7 pathway. GSH is a linear tripeptide and its synthesis process requires two steps. The first step is the rate limiting step, when glutamate and cysteine form #-glutamycysteine with the help of glutamate-cysteine ligase40. GSH synthetase then catalyzes formation of glutathione from #- glutamycysteine and glycine40. In fact, Sappinton et al. demonstrated that significant amounts of

GSH originated from glutamine and that GSH synthesis largely depends on glutamine availability and Gls activity50. Therefore, I speculated that GLS2 supports GSH synthesis cycle by supplying its precursor, GSH. In fact, overexpression of GLS2 by transfecting GLS2 expressing vector increased GSH/GSSG ratio and reduced ROS in the carcinoma cell line51,52. Moreover, silencing

GLS2 on the carcinoma cell line by transfecting GLS2 siRNA oligo reduced GSH/GSSG ratio and increased intracellular ROS level51,52. Thus, GLS2 activity enhances oxidative stress clearance by providing more GSH.

1.7: p53 and GLS2

Several studies claimed that patients with SLE have antibodies against p53, the tumor suppressor protein, indicating that p53 might not be functional in this setting53,54. Moreover, T cell- specific p53 knockout mice spontaneously developed systemic autoimmune disease characterized by systemic lesions involving multiple organs and production of anti-nuclear antibodies and autoantibodies55. These T cell-specific p53 knockout mice also displayed reduced Treg differentiation capacity in vitro compared to the control55. Under normal conditions, p53 has a protective function in maintaining immune homeostasis by augmenting Foxp3 transcription and promoting Tregs55. In addition to its well-known function in tumor suppression, recent studies reported that p53 participates in antioxidant defense. In a carcinoma cell line, p53 directly associates to a response element in the GLS2 promoter region and induction of p53 expression promoted GLS2 expression51,52. Manipulation of p53 expression showed the same trend as GLS2

8 expression alteration: overexpressing p53 decreased cellular oxidative stress and silencing it increased ROS level51,52. Taken together, p53 may support Treg function by promoting GLS2 expression and reducing oxidative stress.

1.8: Research Question

Decreased IL-2 production in humans and mice with SLE is central to the expression of systemic autoimmunity and while clinical trials currently attempt to evaluate the clinical value of administering IL-2 to treat the disease, central questions which pertain to the involved molecular and metabolic mechanisms remain unanswered.

Prior to joining the laboratory, it had been shown that Gls1, the first enzyme in the glutaminolysis pathway, was involved and responsible for the increased production of IL-17 and related autoimmune pathology. Knowing that Gls has two isoenzymes, I asked whether Gls2 was similarly involved in the expression of IL-17. To my surprise, Gls2 was linked not to IL-17 production but rather to the production of the important cytokine IL-2. Searching the literature, I learned that Gls2 clears ROS and that increased ROS level had been linked to decreased IL-2 production.

At that point I formed the hypothesis that decreased GLS2 expression leads to decreased

IL-2 production through distinct metabolic processes. To test my hypothesis, I performed experiments to determine whether GLS2 expression in SLE is low; whether GLS2 limits the production of ROS in T cells; whether restoration of the GLS2 levels would correct IL-2 production.

I believe that full understanding of the GLS2> GSH/GSSG> IL-2 pathway will enrich our therapeutic armamentarium against SLE and other autoimmune diseases.

Chapter 2: Materials and Methods

Mice. MRL/MpJ and MRL/MpJ-Faslpr/J mice aged 15 weeks were used for Western blotting.

C57BL/6J mice aged 8–12 weeks were used for in vitro culture experiments. All mice were purchased from The Jackson Laboratory. All mice were maintained in an SPF animal facility (Beth

Israel Deaconess Medical Center). Experiments were approved by the Institutional Animal Care and Use Committee of BIDMC (IACUC protocol number 088-2015).

Mouse Cell Isolation. Naïve CD4+ T cells (CD4+ CD69L+) were isolated from the spleens of 8-

12 week-old C57BL/6 mice using a magnetic-activated cell sorting (MACS) kit purchased from

Miltenyi Biotec. CD4+ T cells were isolated from other splenocytes by depleting non-CD4+ T cells in the first step. For the second magnetic separation, CD69L+ cells were positively selected.

Human Samples and Human Cell Isolation. Patients who fulfilled the criteria for the diagnosis of SLE as set forth by the American College of Rheumatology56 were enrolled, along with healthy individuals. The BIDMC Institutional Review Board approved the study protocol (2006-P-0298).

Informed consent was obtained from all study subjects. The disease activity for each patient was calculated using the clinico-laboratory index SLE Disease Activity Index57. Age-, sex- and race- matched healthy individuals were chosen as controls (Appendix). Peripheral venous blood of the patients and healthy individuals was collected in heparin-lithium tubes. Peripheral blood mononuclear cell (PBMC) was isolated from peripheral venous blood using lymphocyte separation medium (Mediatech, Inc.) and centrifugation. PBMC was further purified into CD4+ T cells using

CD4+ T Cell Isolation Kit, Human (Miltenyi Biotec). Non-CD4+ T cells were magnetically labelled and depleted during this process.

In-vitro Stimulation for Mouse Cells. The purified naïve CD4+ T cells were plated at 3.0 x 105 cells per well on 48-well flat-bottom culture plates that were previously coated with goat-anti-

10 hamster antibody (2 ng/mL, MP Biomedicals). For Th0 non-polarizing condition, naïve CD4+ T cells were stimulated with soluble anti-mouse CD3 (0.25 μg/mL, Biolegend) and anti-mouse CD28

(0.5 μg/mL, Bioxcell) in a culture media containing RPMI-1640 with 2mM L-glutamine supplemented with 10% fetal bovine serum, 1% penicillin and 0.1% 2-mercaptoethanol. Plate with cells was incubated at 37℃ with 5% CO2.

In-vitro Stimulation for Human Cells. Each well of 48-well flat-bottom plate was coated with

200 μL of 1 μg/μL purified anti-human CD3 (OKT3). The coated plate was incubated at 37℃ with

5% CO2 for at least 2 hours before use. The plate was washed with 200 μL 1X PBS once. Isolated and purified human CD4+ T cells were resuspended in RPMI-1640 with 2mM L-glutamine supplemented with 10% fetal bovine serum, 1% penicillin and soluble 1 μg/mL anti-human CD28.

Final volume was adjusted to 1mL media per well. The plate was centrifuged at 400 rpm for 5 minutes for cell activation and incubated at 37 ℃ with 5% CO2 until analysis. In general, transfected cells were incubated for 2 or 3 days depending on the experiment.

Western Blotting. Reduced and denatured cell lysates were separated on NuPAGE 4-12% Bis-

Tris gel and proteins were transferred to a nitrocellulose membrane. Anti-Gls2, anti-%-actin, goat- anti-mouse IgG couple with horseradish peroxidase (HRP), and goat-anti-rabbit IgG with HRP were used to evaluate the relative level of proteins in each cell lysate sample. Amersham ECL prime system were used for protein detection.

Flow Cytometry. Collected cells were first stained with Zombie Aqua (ZA) Fixable Viability Kit in the dark at room temperature for 15 minutes to distinguish live cells from dead cells. No additional surface staining was performed on mouse cells. For surface staining of human PBMC, cells were incubated with anti-human CD3 PE/Cy7 (UCHT1, Biolegend), anti-human CD4 PerCP- eFlour 710 (SK3, eBioscience), and anti-human CD8a APC (HIT8a, Biolegend) for 15 minutes at

4℃. For detection of intracellular reactive oxygen species, cells were stained with DHR123 (100 ng/mL, Santa Cruz Biotechnology, Inc.) in the dark at room temperature for 30 minutes. For intracellular staining, collected cells were stimulated with aforementioned culture media containing phorbol myristate acetate (500 ng/mL, Sigma-Aldrich), ionomycin (1.4 μg/mL, Sigma-

Aldrich), and golgi stop (1 μL per 1mL culture media, BD Bioscience) for 4 hours.

Cytoperm/cytofix and perm/wash buffer were used to fix and permeabilize the cells stained intracellularly. For mouse cells, anti-mouse IL-2 APC (clone JES6-5H4, Biolegend) was used for intracellular staining. For human cells, anti-human IL-2 PB (clone MQ1-17H12, Biolegend) and anti-human p53 PE (DO-7, Biolegend) were used. All flow cytometry data were obtained with

Beckman Coulter CytoFLEX LX Flow Cytometer and were analyzed with FlowJo v10.5. Cell sorting was performed with BD FACSAria II.

ELISA. IL-2 was measured with sandwich ELISA MAX Deluxe SET Mouse IL-2 and Human IL-

2 (BioLegend). A day before ELISA measurement, a 96-well flat-bottom plate was coated with

100 μL of 1X capture antibody for IL-2 and incubated overnight at 4°C. The plate was washed 4 times with the wash buffer. After washing step, 200 μL 1X assay diluent A was added to each well and the plate was incubated on the shaker at room temperature for 1 hour. The plate was then washed 4 times. 100 μL diluted standards and samples were added to the appropriate well. ELISA plate was incubated for 2 hours at room temperature, shaking. The plate was washed 4 additional times. 100 μL of the diluted detection antibody for IL-2 was added to each well. The plate was incubated at room temperature for 1 hour on the shaker. The plate was washed another 4 times.

100mcl of avidin-HRP solution was added to each well. The plate was incubated for 30 minutes at room temperature on the shaker. The plate was washed 5 times. 100 μL of TMB substrate solution was added to each well. The plate was incubated less than 15 minutes at room temperature

12 in the dark. 100 μL of the stop solution was added to each well. A microplate reader was used to read the absorbance of sample at 450 nm and 570 nm in room temperature. The reading at 570 nm was subtracted from that at 450nm. For mouse IL-2, the standard recombinant IL-2 ranged from

0-125 pg/mL. For human IL-2, the standard IL-2 ranged from 0-500 pg/mL. The sensitivity of the mouse IL-2 assay was 1 pg/mL. Human IL-2 ELISA set can detect IL-2 concentration as low as 4 pg/mL.

Overexpression and Transfection. Mouse cells. Naïve CD4+ T cells were isolated and cultured under Th0 non-polarizing condition. These cells were collected after 24 hours and 7.5-15 μg of empty or overexpression vector was transfected per 1 x 106 cells using the Amaxa Mouse T Cell

Nucleofector Kit with the X-001 program according to the manufacturer’s protocol. The supernatant of each culture was saved during transfection and recovery. After 3 hours of incubation at 37 °C, cells were cultured in the original supernatants for another 24 hours.

Jurkat Cell Line. 2 x 106 cells were electroporated with appropriate plasmid using Amaxa

Nucleofector Kit V with the X-001 program.

Human Cells. CD4+ T cells were isolated from peripheral blood sample as previously described.

7 x 106 cells were used per transfection using Amaxa Human T cell Nucleofector kit with U-014 program.

SiRNA and Transfection for Mouse Cells. The following Silencer Select Pre-Designed siRNA were used: Gls2 siRNA 1, 5’-CUUGCUCUCUGAGACUCAATT-3’; Gls2 siRNA 2, 5’-

UGGUCUGCGCUAUAACAAATT-3’; Silencer Select Negative Control #1 siRNA (Thermo

Fisher Scientific). Naïve CD4+ T cells were isolated and cultured under Th0 non-polarizing condition. 24 hours after seeding, 3 to 30 pmole of siRNA was transfected into each sample using the Amaxa Mouse T Cell Nucleofector Kit with the X-001 program according to the

13 manufacturer’s protocol. The supernatant of each culture was saved during transfection and recovery. After 3 hours of incubation at 37 °C, cells were cultured in the original supernatants for another 24 hours.

Luciferase Assay. Mouse and human IL-2 promoter luciferase reporter constructs on pGL3 background were purchased from Genescript. For mouse cells, Amaxa mouse T cell nucleofector kit and X-001 program were used to transfect luciferase reporter plasmid. For the Jurkat cell line,

Amaxa Cell Line T Cell Nucleofector kit V and X-001 program were used. For human cells,

Amaxa Human T Cell Nucleofector kit and U-014 program were used. 5 μg of IL-2 luciferase reporter plasmid was used for each transfection. As an internal control, 400 ng of renilla luciferase construct was co-transfected to each sample. Dual luciferase reporter assay system (Promega) was used to measure IL-2 production at transcriptional level. All procedures were performed according to the manufacturer’s instructions.

Mutagenesis of Gls2 Promoter Region. Mouse Gls2 promoter luciferase reporter construct

(Mu_promotor_Gls2_pGL3-Basic (U0613BL080-4)) was purchased from Genescript. To mutate potential p53 binding site on Gls2 promoter on mouse cells, the Q5 site-directed mutagenesis kit

(New England Biolabs) and primers mgls2_pGL3_s:5’-GAAGGTCACTACGRGGCTCC-3’ and mgls2_pGL3_As:5’-AGGGGTGGCCAGTGCACA-3’ were used. Sequences of mutagenesis products were checked through Genewiz.

Statistical Analysis. Statistical analyses were performed using GraphPad Prism version 7.0. All statistical significance for human samples was determined by the Mann-Whitney test, unless otherwise indicated. For mouse samples, unpaired Student’s t-test was used. P values < 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

Chapter 3: Results and Discussion

3.1: Results

While investigating the effects of glutaminase on various cytokine production and T cell differentiation, I noticed that Gls2 expression correlated positively with IL-2 production. The Gls2 protein expression level in mice was measured with Western blots and mouse IL-2 production level was measured with ELISA.

GLS2 Expression is Diminished in Patients with SLE.

Many studies have shown that patients with SLE and lupus-prone mice express low levels of IL-233–35. To determine whether there is a correlation between GLS2 and IL-2 expression levels in humans, I performed Western blotting experiments to measure GLS2 and %-actin protein expression levels in patients with SLE and healthy controls. Human CD4+ T cells were isolated from peripheral blood through centrifugation and MACS purification. Then the cells were stimulated on plate-bound human-CD3 (OKT3) and soluble-human-CD28 antibodies overnight.

Stimulated cells were collected and cell lysates were obtained. According to the densitometric analysis of Western blots, patients with SLE had lower GLS2 protein expression compared to their matched controls (Fig. 3.1A,B). 8 healthy donors had a mean GLS2/ %-actin ratio of 0.2631 with

SEM of 0.01573. 8 patients with SLE had mean GLS2/ %-actin ratio of 0.2024 with SEM of

0.0378. The median value of GLS2/ %-actin ratio for healthy donors was 0.2713 and that for patients with SLE was 0.225. When I applied an unpaired t-test, the difference was significant with the p value of 0.0340. However, when I applied the Mann-Whitney test, which is one-tailed non- parametric test commonly used to analyze human samples, the difference was not statistically

15 significant with the p value of 0.1393. Furthermore, data points of patients with SLE were more scattered compared to that of healthy donors.

Gls2 expression is diminished in CD4+ T cells isolated from MRL/MpJ-Faslpr (MRL/lpr), a mouse model for systemic autoimmunity, compared to its control strain, MRL/MpJ.

In order to confirm that Gls2 expression is also diminished in mice prone to lupus, I used

Western blotting to determine Gls2 and %-actin expression level in CD4+ T cells sorted from 15 week-old MRL/MpJ and MRL/lpr mice. CD4+ T cells were sorted from purified splenocytes using

BD FACSAria II. Average Gls2/%-actin ratio for MRL/MpJ was 0.567 with SEM of 0.1408 and that for MRL/lpr was 0.3957 with SEM of 0.008959. Although preliminary, this mouse model of systemic autoimmunity shows the same trend: an SLE disease model has lower Gls2 expression.

Gls2 Overexpression Induces IL-2 Production in Primary Mouse CD4+ T Cell at both the

Protein and Transcriptional levels.

To confirm that Gls2 can modulate IL-2 production at the protein level, empty vector or mGls2-expressing plasmid were transfected into mouse naïve CD4+ T cells and incubated under

Th0-non-polarizing conditions for 1.5 days. Electroporation was used to transfect vectors into cells. 24 hours after transfection, cells were collected and ZA negative and DsRED positive cells were sorted using BD FACSAria II. DsRED is equivalent to PE for flow cytometry reading. ZA negative cells—that is live cells—and DsRED positive—that is successfully transfected cells— were studied. Sorted ZA- DsRED+ cells were permeabilized and fixed with cytoperm/cytofix buffer. The cells were intracellularly stained overnight. The percentage of IL-2 producing cells among live, transfected cells were quantified using flow cytometry (Fig. 3.3B). 89.83% of CD4+

T cells transfected with mGls2-expressing plasmid produced IL-2, whereas 78.25% of CD4+ T

16 cells transfected with empty plasmid produced IL-2 (Fig. 3.3C). Overexpression of mGls2 significantly increased the percentage of IL-2 producing cells with the p value of 0.0024.

To confirm that this finding is consistent at the transcriptional level, IL-2 promoter activity was measured after transfecting cells with empty vector or mGls2-expressing plasmid along with mIL-2 promoter luciferase and renilla. Mouse naïve CD4+ T cells isolated and incubated in Th0- nonpolarizing condition for 1.5 days. IL-2 promoter activity was measured 24 hours post- transfection. Dual luciferase assays showed that the relative IL-2 promoter activity was approximately 2.774 times greater in mouse CD4+ T cells transfected with mGls2-expressing plasmid compared to cells transfected with empty vector (Fig. 3.3D). This difference was statistically significant with the p value of 0.0079.

In order to assess the effects of silencing Gls2 in mouse CD4+ T cells, negative control #1 siRNA or siRNA for mGls2 was transfected to Naïve CD4+ T cells incubated under Th0- nonpolarizing conditions. 3-30 pmol of siRNA was used per each transfection sample. The results were non-conclusive, and the experiment is in the process of further optimization. Incubation conditions and duration of incubation will be modified.

GLS2 Overexpression Increases IL-2 Transcription in Jurkat Cell and Primary Human

CD4+ T Cells.

In order to verify findings in the cell line, the dual luciferase assay was repeated with Jurkat cells. Jurkat cells were transfected with empty vector or hGLS2-expressing vector along with hIL-

2 promoter luciferase and renilla. Transfected Jurkat cells were stimulated with soluble anti-human

CD3 and anti-human CD28 for 2 days. When hIL-2 is transcribed, the luciferase gene is also transcribed, emitting a fluorescent signal. Renilla acts as an internal control that normalizes measurements of gene expression. Although preliminary, the Jurkat cells transfected with hGLS2-

17 expressing plasmid had a relatively greater IL-2 promoter activity. Jurkat cells transfected with hGLS2-expressing vector had 1.981 times higher hIL-2 promoter activity compared to those transfected with empty vector (Fig. 3.4A).

Dual luciferase assay was repeated with primary human CD4+ T cells as well. Human

CD4+ T cells were transfected with appropriate vectors. Transfected human CD4+ T cells were stimulated with plate-bound anti-human CD3 (OKT3) and soluble anti-human CD28 for 2 days.

Collected cells were lysed in passive lysis buffer and stored at -80℃ until analysis. Human CD4+

T cells isolated from healthy donors also showed distinct differences. Human CD4+ T cells transfected with hGLS2-expressing vector transcribed hIL-2 1.936 times more than human CD4+

T cells transfected with empty vector with the p value of 0.05 (Fig 3.4B).

GLS2 Overexpression Reduces Oxidative Stress in Human CD4+ T Cells.

Because GLS2 has been reported to favor GSH over GSSG51,52, I examined the effect of hGLS2 overexpression on ROS generation. Isolated and purified human CD4+ T cells were transfected with empty vector or hGLS2-expressing plasmid via electroporation. Both non- transfected and transfected cells were stimulated in plate-bound anti-human CD3 (OKT3) and soluble anti-human CD28 for 2 days. Cells were collected and stained with ZA and DHR123.

Measurement of ROS levels using DHR123 stain is a simple process. DHR123 is non-fluorescent at baseline. DHR123 can passively diffuse across the membrane. When it interacts with ROS, it becomes oxidized to rhodamine 123 which emits a green fluorescent signal. Cells were gated to isolate the live, ROS positive population (Fig. 3.5A). Non-transfected human CD4+ T cells were used as a control without additional cellular stress (Fig. 3.5B). Transfection was used as source of cellular stress. Transfection increased ROS levels and the shift in histogram was noted. A lower percentage of human CD4+ T cells transfected with hGLS2 were producing ROS (Fig. 3.5C). For

18 example, about 49.05% of empty vector transfected human CD4+ T cells and 26.65% of hGLS2 transfected cells produced ROS. Although preliminary, these results show that GLS2 is involved in oxidative stress regulation and antioxidant defense mechanism. p53 Expression in Patients with SLE is Lower than Healthy Control Subjects.

Because antibodies directed against p53 have been reported in the sera of patients with

SLE and p53 has been reported to serve as a transcriptional enhancer of the Gls2 gene, p53 expression in patients with SLE and healthy controls were measured using flow cytometry. PBMC were isolated from human peripheral blood of patients with SLE and healthy controls. Surface staining was performed using human CD3, human CD4, human CD8 antibodies. Subsequently cells were permeabilized and fixed with cytoperm/cytofix buffer stained intracellularly with a p53 overnight. The percentage of p53-producing cells among live CD4+ T cells (ZA-, CD3+, CD4+,

CD8-) was quantified using flow cytometry (Fig. 3.6A). CD4+ T cells isolated from patients with

SLE produced less p53 than its counterparts (Fig. 3.6B,C). The median percent of p53-producing cells was 93 for healthy donors and 70 for patients with SLE. A Mann Whitney test gave a significant p value of 0.0274 for this difference. However, Mean Fluorescence Intensity (MFI) values were less consistent, giving the p value 0.1642. The median value for MFI was 1172 for healthy donors and 588 for patients with SLE.

A HD SLE HD SLE

GLS2 (66kDa)

!-actin (45kDa)

B GLS2 * 0.4 -actin β 0.2 GLS2/ (densitometry)

0.0

HD SLE

Figure 3.1: GLS2 Expression is Diminished in Patients with SLE. (A) Purified CD4+ T cells isolated from patient with SLE and matched healthy donor were stimulated in plate-bound anti-human CD3 (OKT3) and soluble anti-human CD28 overnight. Healthy donors are age, sex and race matched to patients with SLE. GLS2 and beta actin expression were assessed by Western blotting. Representative data are shown. (B) Densitometry analysis from eight experiments are shown (*P<0.05, mean±s.e.m., two-tailed t-test, n=8).

0.3486

lpr A -Fas

MRL/MpJMRL/MpJ Gls2 (66kDa)

%-actin (45kDa)

Gls2 B 1.0

0.8

0.6 -actin β 0.4 Gls2/ (Densitometry) 0.2

0.0 MRL/MpJ MRL/MpJ-Faslpr

Figure 3.2: Gls2 expression is diminished in CD4+ T cells isolated from MRL/MpJ-Faslpr (MRL/lpr), a mouse model for systemic autoimmunity, compared to its control strain, MRL/MpJ. (A) Purified CD4+ T cells isolated from 15 weeks old MRL/MpJ and MRL/lpr mice were assessed by Western blot for Gls2 and beta actin expression. Representative data are shown. (B) Densitometry analysis from two experiments are shown (mean±s.e.m., two-tailed t-test, n=2).

A B Empty

Lymphocyte Single Cells

A A - - SSC SSC

74.1

FSC-A SSC-H Gls2 ZA

- ZA FSC Single Cells Live Cells

93.7

FSC-Width FSC-Width IL-2

C flow cytometry D IL-2IL-2 reporter reporter activity activity 5 5 * * 100 ** T cells

+ 4 4 90 3 3 80 2 2

70 1 1 Relative Luciferase Activity 60 0Relative Luciferase Activity 0

% of Il-2 producing CD4 Empty Gls2 OE emptyempty Gls2Gls2 OE OE n =n 6= 6 Figure 3.3: Gls2 Overexpression in Primary Mouse CD4+ T Cell Induces IL-2 Production at both the Protein and Transcriptional Levels. (A) Gating strategy to isolate vector transfected, live, single cells for FACS analysis. DsRED positive cells were sorted prior to flow cytometry analysis. (B) Empty vector (Empty) or Gls2-expressing (Gls2) plasmid was transfected to Naïve CD4+ T cells from C57BL/6 mouse that have been incubated in Th0 nonpolarizing condition for 1.5 days. DsRED positive cells were sorted 24 hours post-transfection. The percentage of IL-2-producing cells in those sorted cells were measured by flow p=0.0024 cytometry. (C) Cumulative results of flow cytometry analysis on IL-2 producing CD4+ T cells (**P<0.01, mean±s.e.m., two-tailed t-test, n=6). (D) Cumulative results of IL-2 reporter activity measured by dual- luciferase assay (**P<0.01, mean±s.e.m., two-tailed t-test, n=6).

IL-2 reporter activity A 5

Relative Luciferase Activity 0 empty GLS2 OE n = 2 IL-2 reporter activity B 5 *

Relative Luciferase Activity 0 empty GLS2 OE

Figure 3.4: GLS2 Overexpression Increases IL-2 Transcription in Jurkat Cell and Primary Human CD4+ T Cells. (A) IL-2 promoter activity of Jurkat cell transfected with empty vector (empty) and GLS2- expressing plasmid (GLS2 OE) are shown. After transfection, Jurkat cells were stimulated with soluble anti-human CD3 and anti-human CD28 for 2 days prior to analysis. Each dot represents an independent experiment. (B) IL-2 promoter activity of primary human CD4+ cell isolated from peripheral blood transfected with empty vector (empty) or GLS2-expressing plasmid (GLS2 OE) are shown. After transfection, human CD4+ T cells were stimulated with plate-bound anti-human CD3 and soluble anti- human CD28 antibodies for 2 days prior to analysis (*P<0.05, mean±s.e.m., two-tailed t-test, n=3).

A B

Non-transfected Ctrl A A - -

SSC Lymphocytes SSC Single Cells Mode Empty

GLS2

FSC-A SSC-H ROS C ROS+ Human CD4+ T cells

100

Transfected

80 ZA Live Cells DsRED 60

20 % of ROS producing cells FSC-Width ROS Empty hGLS2

DsRED vector Figure 3.5: GLS2 Overexpression Reduces Oxidative Stress in Human CD4+ T Cells. (A) Gating strategy to isolate transfected, live, single human CD4+ T cells for flow cytometry analysis. (B) Representative histogram of ROS in human CD4+ T cells transfected with empty vector (Empty) or GLS2- expressing plasmid (GLS2). Non-transfected human CD4+ T cells were used as a control. (C) The percentage of ROS producing human CD4+ T cells were quantified by flow cytometry.

B C

Figure 3.6: p53 Expression in Patients with SLE is Lower than Healthy Control Subjects. (A) Gating strategy to isolate single, live CD4+ T cells (ZA-, CD3+, CD4+, CD8-) from PBMC. (B) The percentage of p53-producing cells among CD4+ T cells gated from PBMC of patients with SLE and matched healthy donors were measured by flow cytometry. Healthy donors are age, sex and race matched to patients with SLE (*P<0.05, mean±s.e.m., Mann-Whitney test, n: HC=7; SLE=11). (C) The MFI for p53 expression of human CD4+ T cells are quantified. Performed with help from Dr. Catalina Burbano.

3.2: Discussion

I have demonstrated that Gls2 induces IL-2 production in CD4+ T cells. Because Gls2 is not a transcription factor, I hypothesized that another molecule must be involved in the Gls2- mediated IL-2 induction. Although further experiments need to be conducted to identify the exact mechanism, I suggest that Gls2 activity increases GSH levels in the cells, which normalizes calcium influx to the cell. With normal levels of calcium CREM" remains inactivate and, therefore, does not suppress IL-2 production. In short, I propose Gls2 mediates IL-2 production by reducing intracellular ROS levels via GSH synthesis.

I used Western blotting to determine GLS2 protein level in peripheral CD4+ T cells of humans. Since it is well known that IL-2 level is reduced in patients with SLE and our preliminary data suggest correlation between IL-2 and GLS2, I hypothesized that GLS2 levels will be reduced in patients with SLE compared to their matched control. As expected, there was a trend: patients with SLE generally had lower GLS2 protein levels. To validate this trend, I will continue to analyze more human CD4+ T cells from patients with SLE and their matched control as more samples become available. The average GLS2 expression was lower in patients with SLE, but the range of

GLS2 protein level was greater among patients with SLE compared to their matched controls. This again shows the heterogeneity of the disease. As mentioned earlier, clinical presentations of SLE patients vary among affected patients and, therefore, it is not surprising that patients with SLE display variable GLS2 levels. On the other hand, GLS2 expression among healthy controls was less variable. Even though low GLS2 levels might not represent all the patients affected by SLE, those patients with low GLS2 level would benefit from this study.

Since patients with SLE showed diminished GLS2 expression, I hypothesized that a mouse model of systemic autoimmunity will also produce less Gls2. MRL/lpr is a well-known mouse

26 model for SLE, which spontaneously develops lymphadenopathy and produces autoantibodies58.

MRL/lpr mice have a mutation in lpr that changes Fas receptor transcription, preventing lymphocytes to undergo apoptosis59. MRL/lpr mice were euthanized at week 15 to allow the onset of the systemic autoimmunity. MRL/MpJ mice, which have the control strain for MRL/lpr, were also euthanized at week 15 to match the age. With Western blotting of the sorted CD4+ T cells from MRL/MpJ and MRL/lpr mice, I confirmed that Gls2 protein levels in CD4+ T cells from

MRL/lpr mice are lower compared to cells from control MRL/MpJ mice. Since Gls2 protein is relatively scarce in CD4+ T cells, it was difficult to obtain a good image to compare expression levels. For comparison purposes, sorted CD4+ T cells may be stimulated overnight with anti- mouse CD3 and anti-mouse CD28 prior to collecting cell lysates for Western blotting. In conclusion, Gls2 expression was reduced both in patients with SLE and lupus-prone mice.

I observed that Gls2 overexpression actually induced IL-2 production in mouse CD4+ T cells at the protein and transcriptional levels. When I was first optimizing for vector overexpression experiments, I used 5 'g of vectors per electroporation as suggested by the manufacturer. However, transfection rates were relatively low and, therefore, I decided to increase the amount of vector to

7.5 'g after titration experiment. However, due to the size difference of empty and Gls2-expression vectors, transfection efficiency was different. In general, transfection rates of empty vector were twice as much as that the Gls2-expression vector. Therefore, I chose 7.5 'g of empty vector and

15 'g of Gls2-expression vector as a final amount. With this adjustment, I successfully maintained the transfection rate at approximately 20 percent. Although Gls2 does not directly bind to IL-2 promoter region and initiate transcription, these results confirmed that Gls2 expression is capable of modulating IL-2 levels. Flow cytometry analysis showed increased IL-2 producing CD4+ T cells with Gls2 overexpression vector. Luciferase reporter assays showed that IL-2 transcription

27 rates increased upon Gls2 overexpression. This is significant since IL-2 production is reduced in patients with SLE. If Gls2 can induce IL-2 production, Gls2 might be targeted for therapeutic purposes.

Even though mouse models are commonly used to study human disease, the immune system of the mouse is fundamentally different from that of humans in many ways. Therefore, it is important to establish whether a mouse study is applicable to humans. The GLS2 overexpression experiment was repeated with a Jurkat cell line and human CD4+ T cells. The readings of luciferase assays for IL-2 reporter activity fluctuated slightly from experiment to experiment as

Luciferase Assay Reagent (LAR) II buffer or Stop and Glow buffer was replaced for a new one.

Despite this fluctuation, there was a strong correlation between GLS2 overexpression and hIL-2 transcription. These results indicate that transfecting GLS2-expressing vector to cells clearly increased the transcription of hIL-2.

Measuring IL-2 production at the protein level in the Jurkat cell line and human CD4+ T cells has been challenging. I first attempted to measure IL-2 production with flow cytometry.

Percentages of IL-2-producing CD4+ T cells were scarce and comparable between different conditions. This could be due to high stress caused by electroporation itself. Stressed T cells could be consuming IL-2 to sustain their life. In addition, flow cytometry results are not entirely reflective of how much IL-2 is actually being produced. It is possible that not more cells are producing IL-2, but IL-2 production per cell is increased. To asses to effect of GLS2 overexpression on Jurkat cells and human CD4+ T cells, I am optimizing the conditions to measure hIL-2 by ELISA. Incubating cells in non-polarizing conditions for 24 hours post transfection has been helpful to recover the cells from stress and increase their IL-2 production. I will evaluate

28 more stimulating conditions and time intervals in order to find the ideal conditions and timepoint to compare IL-2 protein production by GLS2-transfected or empty vector-transfected cells.

The GLS2 overexpression experiment clearly shows that GLS2 can induce IL-2 production.

But what is the mechanism behind this? Many recent studies showed that GLS2 feeds the GSH synthesis cycle and, as a result, supports antioxidant defense. Earlier studies on oxidative stress showed that increased oxidative stress affects calcium storage capacity. Increased intracellular calcium levels are known to suppress transcription activity of IL-2. Therefore, I speculated that greater GLS2 activity strengthens antioxidant defense against ROS and this induces IL-2 production. To test this hypothesis, I measured ROS levels after transfecting cells with empty or

GLS2 expressing vectors. Measuring ROS level of vector transfected CD4+ T cells has been challenging. Stimulating cells shortly after transfection probably gave excess stress to these cells and led to high death rates. As a result, ROS level was high among all transfection conditions, making comparison among different conditions difficult. To avoid this problem in the future I will stimulate transfected cells only after allowing them to recover overnight. Although I am still in the process of optimizing the conditions to measure ROS levels in cells, preliminary data suggests that

ROS levels were indeed reduced with overexpression of GLS2. CD4+ T cells seem to clear oxidative stress better after GLS overexpression. Further experiments are required to confirm that

GLS2 supports GSH synthesis and antioxidant defense.

Although I am in the process of confirming, I assume that p53 directly enhances Gls2 expression in T cells. And both p53 level and Gls2 expression levels were diminished in patients with SLE. Considering that one of the hallmarks for SLE is limited IL-2 production2,35,60, I assume that p53 and Gls2 expression is disrupted and this limits IL-2 production in patients with SLE.

Since p53 and Gls2 levels are already low in patients with SLE, recuperating normal expression of those molecules may overcome limited IL-2 production.

In conclusion, I confirmed that Gls2 participates in antioxidant defense and has capacity to induce IL-2 production in CD4+ T cells in vitro. Gls2 is a potential candidate to enhance IL-2 production and support immune homeostasis. Of course, caution must be taken to evaluate therapeutic effects of Gls2 upregulation because SLE is a complex disease and metabolism of immune cells are intertwined together.

Chapter 4: Limitations and Perspectives

Since this project is in the early stages of development, many questions still need to be addressed. A main limitation of this research lies in the fundamental function shared by Gls1 and

Gls2. If both Gls1 and Gls2 convert glutamine into glutamate, the precursor for GSH synthesis, how does Gls2 selectively provide antioxidant defense while Gls1 supports proliferation? Since

Gls1 and Gls2 are regulated by different transcriptional factors, this might be accountable for their opposite effects. Nevertheless, the antioxidant capacity of Gls1 will also need to be evaluated.

Silencing Gls2 expression in mouse naïve CD4+ T cells has been challenging because siRNA transfection is very toxic to the cell itself. Many of the cells died after siRNA transfection.

Moreover, non-specific effects due to siRNA cannot be ignored. In addition, if pre-existing Gls2 protein is sufficient to induce IL-2 at the time of Gls2 siRNA transfection, then silencing Gls2 will not cause any significant change on IL-2 production. Although the Gls2-overexpression experiment already confirmed the relationship between Gls2 expression and IL-2 production, a

Gls2 silencing experiment will strengthen my argument. Therefore, finding an optimal condition to successfully transfect siRNA at high viability is crucial.

Regarding the antioxidant defense function of Gls2, we will confirm the ability of Gls2 to reduce intracellular ROS level in vitro by measuring the GSH/GSSG ratio. If Gls2 over-expression can increase GSH/GSSG ratio in vitro, then we can conclude that Gls2 is clearing ROS by upregulating synthesis of GSH.

p53 binding and regulation of Gls2 gene in T cell will be confirmed by mutating potential binding site of p53 on Gls2 promoter region and measuring the Gls2 promoter activity with dual luciferase assay. Although p53 binding to Gls2 promoter region is established in the carcinoma cell line51,52, its transcriptional activity has not been confirmed in T cells.

p53 expression levels in patients with SLE remains largely controversial. Heterogenous clinical manifestations and the pathogenesis of SLE make molecular characterization more difficult2. Although p53 expression was generally lower in patients with SLE compared to healthy control, the p53 expression levels range was wider for patients with SLE. It will be worth the effort to separate these patients into high- and low-p53 expression cohorts and ask whether p53 level correlates with disease activity at the time of the study. In addition, effector T cell population varies greatly from patients with SLE and healthy control. Therefore, it will be more informative to isolate each effector T cell population and compare phenotypes of those populations directly.

Another caveat to this study is that this project has focused on IL-2 production. Even though IL-2 function and production is crucial in the homeostasis of the immune system, the effects of Gls2 on the production of different cytokines can be measured using multiplex cytokine detection tools. Perhaps effects of Gls2 on IL-2 may modulate other cytokine productions downstream on the pathway.

In order to further identify the molecular mechanism of GLS2 mediated IL-2 induction, a

GLS2 knockout Jurkat cell will be generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. To find a candidate molecule involved, a global metabolite analysis will be performed on a genetically modified Jurkat cell. This metabolite analysis will be useful in identifying the IL-2 regulator involved in this pathway. Since many IL-

2 transcription factors and repressors have been already identified37, observing the level of those molecules will provide deeper insight to the molecular mechanism behind this correlation.

In order to investigate whether Gls2 overexpression can delay or rescue mice from developing systemic autoimmunity in vivo, MRL/lpr mice can be used. To better understand the role of Gls2 in the pathogenesis of autoimmune disease, we will first generate CD4+ T cells

32 constitutively overexpressing Gls2 by using synergistic activator mediator (SAM) technology, a

CRISPR/dCas9-based lentiviral transfection along with guide RNA (gRNA) targeted to Gls2 promoter region61. We will also generate the control CD4+ T cells by the same methodology using non-targeting control gRNA. Then those CD4+ T cells will be transferred to MRL/lpr mice. We will measure serum IL-2 concentration in those mice to assess how the overexpression of Gls2 can affect the disease progression in this in vivo animal model. We speculate that increased IL-2 production by Gls2 overexpression will reduce the disease progression of MRL/lpr mice by expanding Treg population.

Moreover, the functional consequences of upregulated Gls2 can be evaluated by measuring the Treg differentiation capacity. The percentage of Treg will be analyzed after overexpressing

Gls2 in CD4+ T cells and incubating them under Treg polarizing conditions. FoxP3 expression will serve as a surrogate marker. Moreover, in vitro Treg suppression assay should be performed to confirm its suppressive capacity, in addition to FoxP3 expression.

Lastly, once the molecular mechanism of Gls2-mediated IL-2 production is established, the effects of Gls2 on CD8+ T cell population should also be investigated because IL-2 is known to affect CD8+ T cells cytotoxic function23. In fact, cytotoxic CD8+ T cells are dysfunctional in patients with SLE and SLE mouse models62. Moreover, the expanded double negative T cell population seen in SLE is believed to result from downregulation of CD863. Therefore, it will be interesting to explore whether Gls2 influences CD8+ T cells through the same or different mechanisms.

There is increasing interest on the nature of the metabolism checkpoints for immune cell function. Many pathways and regulating mechanisms have been unraveled lately, but there are more to be discovered in this field. Many studies in patients with SLE and mouse models of

33 autoimmune diseases concluded that hypermetabolism of CD4+ T cells is linked to the pathogenesis of SLE. Furthering that investigating, characterizing metabolic abnormalities in SLE

T cells, and identifying potential therapeutic targets will bring us one step closer to targeted, personalized therapy for SLE.

Chapter 5: Bibliography

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Appendix: Demographic Information for Human Subjects

Demographic information for human subjects included in Figure 3.1 is shown.

SLE Age (year) Sex Race SLEDAI

n=8 mean±median mean±median 36.25±0.75 3.25±2.25 Lupus1 25 F Asian 0 Lupus2 24 F Asian 0 Lupus3 33 F White 0 Lupus4 44 F Mixed (Black/White) 2 Lupus5 47 F White 14 Lupus6 27 F White 0 Lupus7 52 F White 4 Lupus8 38 F Black 6

Healthy Donor Age (year) Sex Race

n=8 mean±median 36.5±1.5 HD1 25 F Asian HD2 23 F Asian HD3 37 F White HD4 48 F Black HD5 42 F White HD6 27 F White HD7 51 F White HD8 39 F Black