Decreased GZMB, NRP1, ITPR1, and SERPINB9 Transcripts Lead to Reduced Regulatory T Cells Suppressive Capacity in Generalized Vitiligo Patients

Generalized vitiligo (GV) is an autoimmune skin disease characterized by bilateral white patches over the entire body. Regulatory T cells (Tregs) maintain peripheral tolerance; however, they are found to be reduced in numbers and function in vitiligo patients. The exact mechanism for reduced Treg suppressive capacity is unknown. Therefore, we aimed to assess transcript levels of Tregs-associated immunosuppressive genes (GZMB, NRP1, PDCD1, FASLG, and TNFRS18), regulatory molecules of Tregs suppressive function (SERPINB9, ITPR1, and UBASH3A), and Treg-associated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) in 52 GV patients and 48 controls by real-time PCR (qPCR). We found significantly reduced GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs compared to controls (p = 0.03, p = 0.023, p = 0.0045, and p < 0.0001, respectively). There were 0.44-, 0.45-, 0.32-, and 0.54-fold decrease in GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs. Additionally, disease activity and severity-based analyses revealed significantly decreased GZMB (p = 0.019 and 0.034), SERPINB9 (p = 0.031 and p = 0.035), and ITPR1 (p = 0.0003 and p = 0.034) transcripts in active vitiligo and severe GV patients' Tregs. Interestingly, we found a positive correlation for ITPR1 with GZMB (r = 0.45, p = 0.0009) and SERPINB9 (r = 0.52, p = 0.001) transcripts in GV Tregs. Moreover, we found positive correlation for percentage Treg mediated suppression of CD4+ and CD8+T cells with ITPR1 (r = 0.54; r = 0.49), GZMB (r = 0.61; r = 0.58), NRP1 (r = 0.55; r = 0.52), and SERPINB9 (r = 0.56; r = 0.48) in GV Tregs. Further, calcium treatment of Tregs resulted into significantly increased ITPR1, SERPINB9, and GZMB transcripts in GV Tregs (p = 0.023, p = 0.0345, p = 0.02). Overall, our results for the first time revealed the crucial role of GZMB, NRP1, SERPINB9, and ITPR1 transcripts in decreased Treg suppressive capacity leading to GV pathogenesis, progression, and severity. In addition, our study highlighted that ITPR1 might be linked with decreased GZMB and NRP1 expression in GV Tregs. Moreover, our study for the first time suggest that increased SERPINB9 transcripts may lead to endogenous granzyme B-mediated Tregs apoptosis, and calcium treatment of Tregs may improve the Treg suppressive capacity. These findings may further aid in development of Treg-based therapeutics for GV.


Introduction
Generalized vitiligo (GV) is an autoimmune disease characterized by symmetrical white patches on the entire body [1]. Its prevalence is about 0.5 to 2% worldwide [2]. The key role of autoimmunity in GV has been suggested by the presence of autoantibodies and autoreactive CD4 + and CD8 + T cells in vitiligo skin lesions [3]. Furthermore, if they remain unchecked, these autoreactive T cells can lead to granzyme and FAS-FASL-mediated melanocyte destruction, leading to GV pathogenesis [4][5][6][7][8]. Additionally, our previous studies have found the role of inflammatory cytokines IFN-γ and TNF-α in melanocyte destruction [6,[9][10][11].
Therefore, to delineate the exact pathway of Treg cells dysfunction, the current study aimed to study the mRNA expression levels of (i) Tregs-associated immunosuppressive genes (GZMB, NRP1, PDCD1, FASLG, and TNFRS18), (ii) regulatory molecules of Tregs function (SERPINB9, ITPR1, and UBASH3A), and (iii) Treg associated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) in GV pathogenesis, progression, and severity. Additionally, we carried out the age of onset and gender-based analysis for these genes to study their effect on age of onset and gender biasness for GV pathogenesis.

Study Population.
A total of 52 GV patients and 48 healthy controls were included in the study. Table 1 depicts the demographic details for the enrolled participants. Vitiligo was diagnosed by a dermatologist from Aura skin care clinic, Vyara, India, by observing symmetrical white color lesions on skin under woods lamps. Newborn babies, pregnant/lactating women, patients on treatment, and patients with other autoimmune diseases were excluded from the study. Controls were free from any signs of vitiligo and other autoimmune diseases. The study protocol was per the Institutional Human Research Ethics Committee (IHREC), Maliba Pharmacy College, UKA Tarsadia University, India. The study protocol followed the Helsinki Declaration of 1964 and its successful amendments. The GV patients were divided into active vitiligo (AV) patients and stable vitiligo (SV) patients as mentioned previously [15]. The patients were categorized as AV, if they developed any increase in lesions size or number within the past six months; otherwise, they were categorized as SV [28]. Moreover, patients were categorized based on the disease severity measured by the vitiligo area scoring index (VASI) as described by Bhor and Pande [29]. GV patients were divided into three groups: (i) 10%-25% VASI: mild GV; (ii) 25%-50% VASI: moderate GV; and (iii) 50%-75%: severe GV, as mentioned previously [6].
2.2. Isolation of CD4 + CD25 + Treg Cells and CD4 + T Cells. CD4 + CD25 + Treg cells and CD4 + T cells were isolated from three-milliliter peripheral blood of 52 GV patients and 48 controls using MACSxpress® whole blood Treg isolation kit (Miltenyi Biotec, Auburn, CA) as mentioned previously [15]. In the first step, all the non-CD4 + cells were removed from the whole blood using MACSxpress beads by negative selection. In the second step, through positive selection, the CD4 + CD25 + Treg cells were enriched from CD4 + T cells using microbeads and LS columns under a strong magnetic field. Flow cytometry was carried out to confirm the purity of isolated Treg cells (Figure 1), the purity of isolated CD3 +-CD4 + CD25 + FOXP3 + Treg cells was found to be 94.22%. The isolated Treg cells were immediately processed for the downstream experiments.
2.3. Isolation of CD8 + T Cells. CD8 + T cells were isolated from two-milliliter blood sample of GV patients and controls using MACSxpress® whole blood CD8 T cell isolation kit human (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions and as mentioned previously [6]. All the non-CD8 T cells were immunomagnetically depleted with MACSxpress beads. The isolated CD8 + T cells were immediately processed for in vitro Treg suppression assay.
2.4. Total RNA Isolation and cDNA Synthesis. The total RNA was extracted from CD4 + CD25 + Treg cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as mentioned previously [15]. RNA integrity, yield, and purity were determined by 1.5% gel electrophoresis and spectrophotometrically at 260/280 nm. The cDNA was synthesized from 1 μg of total RNA by iScript™ cDNA Synthesis Kit (Bio-Rad, CA, USA) as per the manufacturer's instructions.

Quantitative
Real-Time PCR . The mRNA expression  levels of Treg-associated genes GZMB, NRP1, ITPR1, SER-PINB9, PDCD1, FASLG, UBASH3A, IKZF4, GATA2,  GATA3, TNFRSF18, RUNX1, STAT3, and STAT5 were measured with qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression levels were used as a reference gene. Gene-specific primers for the expression study are mentioned in Table S1. mRNA expression analysis was carried out using iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA) as per the manufacturer's instructions. The qPCR conditions for the gene expressions study are mentioned in Table S1. The specificity of the qPCR products was checked by dissociation curve analysis ( Figures S1 and S2). The fluorescence data were collected during the extension step and the cycle at which the fluorescence intensity raised above the background was termed as cycle threshold (C T ). The difference between the target and reference genes C T was considered as ΔC T value. The ΔΔC T value was determined as the difference between the ΔC T value of controls and patients. The fold change value in gene expression was calculated using 2 -ΔΔCT formula.
2.6. In Vitro Calcium Treatment of Treg Cells. Treg cells were subjected to calcium treatment by dissolving 750 μM calcium (Cayman, MI, USA) in RPMI media. The dissolved calcium level in the medium was confirmed using calcium assay kit (Cayman, MI, USA). Treg cells were seeded in 24-well plate at density of 5 × 10 4 cells, in 1 ml RPMI medium supplemented with 5% FBS containing desired calcium concentration at 37°C at 5% CO 2 for 24 hours. The calcium-treated Treg cells were immediately processed for downstream in vitro functional assays. The standard curve for the estimation of calcium is presented in Figure S3.

2.7.
In Vitro Treg Suppression Assay. CD4 + CD25 + Treg cells (5000 cells) were co-cultured with CD8 + T cells and CD4 + T cells (10,000 cells) at a ratio of 1 : 2 individually, as mentioned previously [6]. The cells were activated with 200 IU rIL2 (PeproTech, NJ, USA) and anti-CD3/CD28 dynabeads Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a 1 : 1 (bead:cells) ratio in 200 μl RPMI supplemented with 10% fetal bovine serum for 5 days at 37°C and 5% CO 2 in 96 well U-bottom plate. On 4th day, the cells were labelled with 10 μM BrdU (Sigma-Aldrich, MO, USA) and incubated for 18 hrs and then further processed for BrdU cell proliferation assay as mentioned previously [6,30,31]. 2.9. Correlation of ITPR1, GZMB, NRP1, and SERPINB9 Transcripts with In Vitro Treg Suppressive Capacity. The isolated Tregs population was divided into two fractions. The inherent levels of ITPR1, GZMB, NRP1, and SERPINB9 transcripts were assessed from the first fraction, whereas in vitro Treg suppression assay was carried out from the second Treg fraction. Further, the inherent levels of ITPR1, GZMB, NRP1, and SERPINB9 transcripts in Tregs of GV were correlated with the Treg suppressive capacity by Spearman's rank correlation analysis.
2.10. Statistical Analysis. The comparison of mean ΔC T values in GV patients and controls for relative mRNA expression analysis, disease activity analysis, disease severity analysis, age of onset analysis, and gender-based analysis was carried out using nonparametric Mann-Whitney U test. The 2 -ΔΔCT analysis was carried out to calculate the fold difference in gene expression. The statistical analysis was carried out using GraphPad prism software (Graphpad software

Transcript Levels of Tregs Associated Immunosuppressive
Genes (GZMB, NRP1, PDCD1, FASLG, and TNFRS18) in GV Patients and Controls. The transcript levels of Tregsassociated immunosuppressive genes (GZMB, NRP1, PDCD1, FASLG, and TNFRS18) were assessed in 52 GV patients and 48 controls using nonparametric Mann-Whitney U test after normalization with GAPDH expression. We found significantly reduced transcript levels for GZMB and NRP1 expression in GV Tregs compared to control Tregs (p = 0:03 and p = 0:023; Figures 2(a) and 2(d)). The 2 -ΔΔCT analysis suggested a 0.44-and 0.45-fold difference in mRNA expression levels of GZMB and NRP1 in GV Tregs compared to control Tregs (Figures 2(c) and 2(e)). Further, the disease activity and disease severity-based analysis suggested significantly reduced mRNA expression levels of GZMB in AV and severe GV Tregs compared to SV and mild GV Tregs (p = 0:019 and p = 0:034, respectively; Figures 2(a) and 2(b)). However, there was no significant difference in mRNA expression levels of NRP1 in AV and severe GV Tregs compared to SV and mild GV Tregs (p = 0:453 and p = 0:2642; Figures 2(d) and 2(e)). Additionally, we did not find any significant difference in transcripts levels of PDCD1, FASLG, and TNFRS18 in GV Tregs compared to control Tregs (p > 0:05; Figure S4). Moreover, the disease activity and disease severity-based analysis suggested no significant difference in transcripts levels of PDCD1, FASLG, and TNFRS18 in AV and severe GV Tregs compared to SV and mild GV Tregs (p > 0:05; Figure S4). ) as compared to SV and mild GV Tregs. However, we could not find any significant difference in transcript levels of UBASH3A for GV Tregs as compared to controls' Tregs (p = 0:145; Figure S5). The disease activity and severity-based analysis also suggested no significant difference in mRNA expression levels of UBASH3A in AV and severe GV Tregs compared to SV and mild GV Tregs (p = 0:487; Figure S5).

Transcripts Levels of Treg Associated Transcription
Factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) in GV Patients and Controls. The transcript levels for Tregassociated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) were assessed in 52 GV patients and 48 controls using nonparametric Mann-Whitney U test after normalization with GAPDH expression. We did not find any significant difference for transcript levels of GATA2, GATA3, RUNX1, STAT3, and STAT5 in GV Tregs when compared to controls' Tregs (p > 0:05; Figures S5 and S6). Moreover, the disease activity and severity-based analysis also suggested no significant difference in mRNA expression levels of GATA2, GATA3, RUNX1, STAT3, and STAT5 in AV and severe GV Tregs as compared to SV and mild GV Tregs (p > 0:05; Figures S5 and S6).

Discussion
Autoimmunity has been strongly implicated in GV pathogenesis by the presence of autoantibodies and autoreactive CD4 + and CD8 + T cells in GV patients [3][4][5][6][7][8]. Additionally, studies have implicated the role of cytotoxic T cells in melanocyte death in vitiligo patients [7,8,15]. Tregs control such autoimmune responses against melanocytes by actively suppressing self-reactive T cells activation and expansion [13,14]. However, studies have suggested impaired Tregs' number and Tregs suppressive function in GV patients [6,14,15]. Moreover, our recent study has suggested the role of altered expression of NFATs and FOXP3 in impaired Treg suppressive function [6]. Thus, impaired Tregs fail to control the ongoing autoimmune response leading to widespread self-reactive T cells activation, resulting in GV pathogenesis [6]. Overall, these studies highlight that Tregs and their suppressive molecules may represent a potential therapeutic target for developing Treg-based therapeutics for GV. Inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) governs the release of calcium from the endoplasmic reticulum [22]. The exact role of ITPR1 in Treg function in GV is unknown. However, upon TCR stimulations in T cells, ITPR1 controls the release of stored calcium from the endoplasmic reticulum [33]. Our previous study has suggested that optimal calcium levels in Tregs are a prerequisite for NFATc1 activation in Treg of GV patients [32]. In current study, we found significantly reduced ITPR1 transcripts in GV patients. Moreover, our study suggested the role of decreased ITPR1 transcripts in GV progression and disease severity. Thus, the reduced ITPR1 expression in Tregs may lead to reduced intracellular Treg calcium levels, resulting in impaired NFAT signalling pathway ( Figure 6). Finally, the impaired NFAT signalling pathway may lead to decreased downstream immunosuppressive molecules leading to impaired Treg suppressive function.
As altered ITPR1 transcripts may lead to impaired Treg activation and suppressive function, we assessed the mRNA expression of Treg-associated suppressive molecules GZMB,  Studies have suggested that Tregs control the immune response by granzyme B-dependent cytotoxicity [34]. Upon TCR stimulation and receptor activation, Treg cells produce granzyme B, and previous studies have found increased CD107a expression upon Treg activation, suggesting extracellular degranulation of granzyme B [21,35]. However, our study revealed significantly reduced GZMB transcripts in GV patients. Moreover, we found that the reduced GZMB transcripts were associated with GV disease severity and disease progression, suggesting the crucial role of GZMB expression in Tregs function. Furthermore, we found a positive correlation between GZMB transcripts and ITPR1 transcripts. Therefore, our study suggests that impaired ITPR1 transcripts might lead to reduced Treg intracellular calcium levels leading to impaired NFAT signalling pathway in GV ( Figure 6). Additionally, our previous study has suggested altered calcium NFAT signalling pathway in GV Tregs [36]. Although the role of NFAT signalling pathway in granzyme B production is unknown, interestingly, studies have suggested for NFAT-binding sites on GZMB promoter [36]. Therefore, the decreased ITPR1 transcripts could further lead to impaired calcium-NFAT signalling pathway, resulting in decreased GZMB transcripts, and thus lead to impaired Tregs suppressive function in GV ( Figure 6). However, future studies must explore the role of ITPR1 and NFAT signalling pathway in granzyme B expression. Furthermore, single-nucleotide polymorphisms (SNPs) and epigenetic changes in GZMB promoter must be explored as they might be responsible for the decreased GZMB expression.
Next, we evaluated the expression levels of Treg suppressive molecule neuropolin 1 (NRP-1). NRP-1 is constitutively expressed on the surface of Tregs and mediates prolonged binding with immature dendritic cells [19,37]. Anti-NRP-1 antibodies have been shown to abrogate Treg immunosuppressive function [38]. Additionally, lack of NRP-1 on surface of Tregs has been linked with impaired Treg suppressive function and worsening of experimental autoimmune encephalomyelitis severity [38]. However, the role of NRP-1 in GV is unknown. Interestingly, we found significantly reduced NRP1 transcripts in GV patients, suggesting for the crucial role of NRP1 in reduced Treg suppressive function leading to GV pathogenesis. Moreover, we found a positive correlation of NRP1 with ITPR1 transcripts. Although the link between ITPR1-calcium-NFAT signalling pathway and NRP1 is unknown, previous studies suggest that NRP1 expression is accompanied with high levels of NFATc1 transcript expression [39]. Additionally, NRP-1 expression is controlled by T cells activation, and inhibition of NFATs has shown to suppress NRP-1 expression in Tregs [39]. Overall, our results suggest that the altered ITPR1-calcium-NFAT signalling pathway might be involved in reduced NRP1 transcripts which led to impaired Treg suppressive function in GV patients ( Figure 6). However, future studies must explore the involvement of ITPR1-calcium-NFAT signalling pathway, promoter SNPs, and epigenetic changes in NRP1 promoter, as they might be responsible for decreased NRP1 expression.
Furthermore, we assessed the expression levels of SER-PINB9 in GV Tregs. SERPINB9 is an endogenous granzyme B inhibitor [21,40]. After Treg activation, there is an increase in the production of granzyme B and its 11 Journal of Immunology Research endogenous inhibitor SERPINB9 [21]. Previous studies have shown an increase in granzyme B-mediated apoptosis in SERPINB9 knockout mice, suggesting that Tregs upon activation can undergo self-inflicted apoptosis mediated by granzyme B in absence of SERPINB9 [21]. Interestingly, our study suggested significantly reduced SERPINB9 transcripts in GV Tregs. Moreover, we found a significant association of SERPINB9 transcripts with GV disease severity and disease progression. Additionally, previous studies have suggested significantly decreased Treg cells in vitiligo patients [6,14,15], suggesting that the increased SERPINB9 transcripts can lead to Treg cells apoptosis, resulting into decreased Tregs number, thereby contributing to GV pathogenesis, progression, and severity.  Figure 6: Role of GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV pathogenesis. (a) The decreased ITPR1 transcripts could lead to impaired calcium-NFAT signalling pathway, which might result in decreased GZMB and NRP1 transcripts. Further, the decreased SERPINB9 transcripts may result in increased granzyme B-mediated endogenous apoptosis of Tregs. Overall, the decreased GZMB, NRP1, SERPINB9, and ITPR1 transcripts result into decreased Treg suppressive capacity, which could lead to unchecked CD4 + and CD8 + T cells and thereby results into melanocytes' destruction contributing to GV pathogenesis, progression, and severity. (b) Upon calcium treatment, ITPR1 mRNA expression is increased which may lead to intracellular Treg calcium influx and calcium-NFAT signalling pathway, thereby results into increased GZMB and SERPINB9 transcripts, leading to increased Treg suppressive capacity. The increased Treg suppressive capacity controls the CD8 + and CD4 + T cells proliferation and IFN-γ production and thereby contributes to melanocytes survival.
However, future studies must confirm these findings by studying granzyme caspases-mediated apoptosis pathway in Tregs of GV patients. Next, we aimed to study the key Treg-associated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) in GV patients. The GATA transcription factors are zinc finger motif DNA-binding proteins, and they have a crucial role in Treg function [26,27]. Genetic ablation in GATA3 has been associated with inflammatory disorder in mice [26]. Additionally, mutations in GATA2 have been associated with autoimmune hepatitis [27]. Additionally, RUNX1 is a runt-related transcription factor, and it plays a crucial role in generation and function of Treg cells [25]. Moreover, STAT transcription factors, i.e., signal transducers of activation of transcription, control Treg cells development [24,41]. Genetic knockdown of STAT3 decreases the Tregs generation [24]. Moreover, STAT5 plays a crucial role in sustaining FOXP3 expression in Tregs [41]. However, we did not find an association for Treg-associated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) with GV pathogenesis, progression, and severity.
Previously, we had studied the in vitro Treg suppression assay in GV patients [6]. Our study had revealed significantly decreased in vitro Treg-mediated suppression of CD4 + and CD8 + T cells in GV patients [6]. To study the role of ITPR1, GZMB, NRP1, and SERPINB9 on in vitro Treg suppressive capacity, we carried out correlation analysis. Interestingly, we found a positive correlation for Treg-mediated suppression of CD4 + and CD8 + T cells with ITPR1, GZMB, NRP1, and SERPINB9 transcripts in GV patients' Tregs. Therefore, our results suggest that decreased ITPR1, GZMB, NRP1, and SERPINB9 transcripts might impair Treg suppressive function, resulting in widespread activation of CD4 + and CD8 + T cells, which could lead to GV pathogenesis ( Figure 6).
Our previous study suggested that calcium treatment of Tregs increased the intracellular calcium influx in Tregs of GV patients, due to increased expression of calcium ion channel gene ORAI1 after calcium treatment [42]. Moreover, the optimum calcium influx enhanced the calcineurin and NFATc1 activity in calcium treated Tregs, which led to increased Treg suppressive capacity [32]. As ITPR1 is crucial for the release of calcium in Tregs, we studied the transcript levels of ITPR1 in calcium-treated Tregs of GV patients and controls. Interestingly, our study suggested an increase in mRNA expression of ITPR1 in Tregs after the calcium treatment (Figure 3(d)). As the increased expression of ITPR1 after the calcium treatment may further lead to enhanced Treg suppressive activity, we accessed the transcript levels of GZMB and SERPINB9 in calcium-treated Tregs of GV patients. Our study suggested that the calcium treatment increased the expression levels of GZMB and SERPINB9 in GV Tregs (Figures 2(a) and  3(a)). Our results are in line with the previous findings which suggest that calcium signalling modulates cytolytic T lymphocyte function [42]. Overall, our study suggests that calcium treatment may improve Treg suppressive capacity, and targeting the Ca 2+ -calmodulin-calcineurin-NFAT signalling pathway may be a potent therapeutic target for GV.

Conclusions
Overall, our results for the first time suggest the crucial involvement of GZMB, NRP1, SERPINB9, and ITPR1 transcripts in reduced Treg-mediated suppression of CD4 + and CD8 + T cells which lead to GV pathogenesis, progression, and severity. Moreover, our study highlighted that ITPR1 might be responsible for decreased GZMB and NRP1 transcripts in GV Tregs. Furthermore, our study revealed that the increased SERPINB9 transcripts might result in endogenous granzyme B-mediated Tregs apoptosis, and the Treg suppressive capacity can be enhanced after calcium treatment in Tregs. These findings may aid in development of Treg-based therapeutics for GV; however, in vivo studies must be carried out to validate the role of GZMB, NRP1, SERPINB9, and ITPR1 in Treg-mediated GV pathogenesis.

Data Availability
Data available on request.

Ethical Approval
All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institutional-Human Research Ethical Committee (IHREC), Maliba Pharmacy College, Uka Tarsadia University, Bardoli, Gujarat, India, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All patients and healthy control subjects signed informed consent.

Consent
Informed consent was obtained from all patients and control participants of the study.

Conflicts of Interest
We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.

Authors' Contributions
Prashant S. Giri has contributed to the investigation, methodology, validation, analysis, and writing-original draft preparation. Ankit H Bhart has contributed to the resources (patients' samples), clinical information, and analysis. Mitesh Dwivedi has contributed to the conceptualization, funding acquisition, investigation, methodology, resources, supervision, visualization, and writing-reviewing and editing.

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Journal of Immunology Research