Table 1.

Main clinical studies including information on DNT cells in patients with SLE.

Authors, Year, Country	Study Design	Primary Study Aim	SLE Pts. (n)	SLE pts. (Gender, Age)	SLE pts. Disease Duration	SLE Groups	SLE Groups (n, Age)	SLE Groups’ Disease Duration	Controls [n; Gender; Age]	DNT Cells Immuno-phenotype	DNT Cells [% CD3+]	Flow Cytometry Equipment	Therapy	DNT Cell-Related Findings
Shivakumar et al., 1989, USA [24]	Prospective Cross- sectional	- To investigate the production of cationic anti-DNA IgG in lupus nephritis and cellular mechanisms regulating this process.	20	M:F = 3:18 Age n/a	n/a	- Active (with nephritis) - Inactive	n = 12 range 22–34 yrs. n = 8 range 35–55 yrs.	range 0.5–12 yrs. range 8–12 yrs.	n = 8 M:F = 3:5 range 20–30 yrs.	CD3+ CD4− CD8− TCRαβ+	Mean ± SD Active: 2.7 ± 0.80 Inactive: 0.9 ± 0.06 Controls: 0.27 ± 0.09 I vs. C [p < 0.001] A vs. C [p< 0.001] A vs. I [p< 0.001]	FACScan	n/a	- DNT cells were markedly expanded in SLE patients and, along with CD4+T cells, supported the production of pathogenic anti-DNA IgG with cationic charge.
Liu et al., 1998, Taiwan [33]	Prospective Cross- sectional	- To investigate DNT cells in the peripheral blood mononuclear cells of SLE patients.	47	M:F = 4:43 Mean (range) 30 yrs. (12.0–58.0)	n/a	- Active (with nephritis) -Inactive	n = 26 n = 21	n/a n/a	n = 44 M:F = 3:41 “Similar age”	CD3+ CD4− CD8− TCRαβ+	Mean ± SD SLE: 1.14 ± 0.88 Controls: 0.88 ± 0.54	FACSsort	- “Majority of patients were taking variable doses of steroids”. - Cytotoxic drugs (n = 21)	- Increased number of DNT cells was found in SLE patients, but neither association with lupus nephritis nor correlation with disease activity and anti-DNA titers was observed.
Sieling et al., 2000, USA [34]	Prospective Cross- sectional	- To investigate DNT cells and mechanisms leading to IgG autoantibody production in SLE.	20	M:F = 2:18 Mean (range) 39.1 yrs. (13–68)	Mean 8.7 yrs (0.5–25)	-	-	-	Yes (n = n/a) F = 57% Mean 32 yrs.	CD3+ CD4− CD8− TCRαβ+	Mean ± SD SLE: 3.0 ± 0.4 Matched donors: 0.6 ± 0.1 Unmatched donors 1.0 ± 0.2 [p <0.005]	n/a	Prednisone (0–40 mg/die) Cytotoxic drugs (n = 6)	- DNT cells from SLE patients produced both IL-4 and IFN-γ and supported CD1c1+ B cells to produce IgG antibodies.
Dean et al., 2002, UK [39]	Prospective Cross- sectional	- To assess the percentage of IL-4+ DNT cells from patients with SLE and compare them with conventional T lymphocytes.	50	M:F = 1:49 Mean (range) 37.2 yrs. (17–66)	n/a	Variable disease activity	-	-	n = 16 M:F = 3:13 Mean (range) 36.1 yrs. (21–57)	CD3+ CD4− CD8− TCRαβ+	n/a $	FACScan	- SLE patients were on steroid and/or Immuno- suppressive drugs, but no detailed information.	- IL-4+DNT cells were more frequent in peripheral blood of patients with SLE than healthy controls.
Crispin et al., 2008, USA [35]	Prospective Cross- sectional	- To investigate DNT cells and their cytokine production in patients with SLE.	24	M:F = 0:24 Mean (range) 40.2 yrs. (25–57)	n/a	Variable disease activity	-	-	n = 16 n/a	CD3+ CD4− CD8− TCRαβ+	n/a $	FACSAria	“Prednisone was discontinued at least 24 h before venipuncture”.	- DNT cells from SLE patients can produce IL-17 and IFN-γ. In detail, IL-17 producing cells and DNT cells are present in kidney biopsies of SLE patients.
Lai et al., 2012, USA [36]	Prospective, Controlled, Double-blind trial	- To assess the safety, tolerance, and efficacy of the GSH precursor NAC and its related immunobiologcal impact.	36	M:F = 2:34 Mean ± SEM (range) 44.6 ± 1.8 yrs. (25–64)	n/a	Inactive (or stable disease)	-	-	n = 42 M:F = 3:39 Mean ± SEM (range) 44.4 ± 1.7 yrs. (22–63)	CD3+ CD4− CD8−	Mean ± SD Baseline: 6.2 ± 0.5 After 3-mo NAC: 5.3 ± 0.5 [p = 0.043]	n/a	n/a	- “The mean±SEM 1.35± 0.12-fold DNT cells in patients with SLE compared to matched healthy controls (p = 0.008) was eliminated by NAC treatment, which also increased the mitochondrial hyper-polarization, mass, and apoptosis of DNT cells in SLE patients”.
Lai et al., 2013, USA [37]	Prospective Longitudinal	- To assess the mitochondrial dysfunction and mTOR activation in peripheral blood mononuclear cells from SLE patients.	59	M:F = 3:56 Mean ± SEM (range) 43.1 ± 1.6 yrs. (20–65)	n/a	-	-	-	n = 54 M:F = 7:47 Mean ± SEM (range) 39.1 ± 1.8 yrs. (20–62)	CD3+ CD4− CD8−	n/a $	n/a	n/a	- mTOR activation increases the production of IL-4 and necrosis of CD3+/CD42/ CD82 DNT cells.
Tarbox et al., 2014, USA [41]	Prospective Cross- sectional	- To assess DNT cells in several pediatric autoimmune diseases, including SLE.	23	M:F = 5:18 Mean ± SD (range) 13 ± 5 yrs. (2–25)	n/a	-	-	-	n = 28 M:F = 7:21 Mean ± SD (range) 17 ± 5 yrs. (7–25)	CD3+ CD56− CD4− CD8− TCRαβ+ TCRγδ−	Mean ± SD (range) SLE: 2.2 ± 0.9 (0.4–4.5)	n/a	- No cytotoxic drugs (n = 19) - Cytotoxic drugs (n = 17) - Steroids only (n = 3) - Steroids + cytotoxic drug (n = 15)	- A portion (34.8%, slightly higher than other rheumatic disease, but not significantly) of SLE patients showed increased number of DNT cells. In general, patients with increased DNT cell percentages showed increased CD45RA expression.
Wang et al., 2014, China [42]	Prospective Cross- sectional	- To assess DNT cells, their Fas expression, and intracellular cytokine levels in SLE patients.	120	M:F = 9:111 Mean ± SEM (range) 29.6 ± 1.1 yrs. (9–63)	n/a	- Active - Inactive	n = 82 n = 38	n/a n/a	n = 43 M:F = 3:40 Mean ± SEM (range) 30.6 ± 1.4 yrs. (7–25)	CD3+ CD4− CD8− TCRαβ+	Mean ± SEM SLE: 2.32 ± 0.12 Active: 2.68 ± 0.16 Inactive: 1.55 ± 0.11 Control: 1.03 ± 0.09 I vs. C [p < 0.001] A vs. C [p < 0.001] A vs. I [p < 0.001]	FACS Calibur	n/a	- DNT cells are increased in SLE patients and their value positively correlated with disease activity. - Abnormal Fas expression was observed in DNT cells.
El Sayed et al., 2017, Egypt [43]	Prospective Longitudinal	- To assess peripheral DNT cells in pediatric SLE and their potential correlation with disease activity and different organ damage.	21	M:F = 0:21 Mean ± SD (range) 13 ± 2 yrs. (10–17)	n/a	- new diagnosis (active) - previous diagnosis (active)	n = 12 n = 9	0 yrs. (diagnosis) range 0.5–3 yrs.	n = 20 M:F = 0:20 Mean ± SD (range) 14 ± 2 yrs. [11,12,13,14,15,16,17]	CD3+ CD4− CD8− TCRαβ+	Median (IQR) Disease activity: 3.7 (3.0–5.7) Disease remission: 1.4 (1.2–1.8) Controls: 1.0 (0.5–1.4) Active New SLE: 5.0 (3.7–5.9) Active Old SLE: 2.8 (1.7–3.4)	Epics XLTM Navios	- “All patients received corticosteroid treatment during the period of follow-up” - CPM (n = 7) - MMF (n = 7) - Rituximab (n = 3)	- DNT cell percentage was significantly higher in proliferative nephritis than in non-proliferative nephritis but was comparable between patients with and without nephritis. - Active patients had more frequent DNT cell increase than those in remission. - DNT cell percentages showed a significant and positive correlation with SLEDAI-2K score and were higher in newly diagnosed SLE patients.
Stratigou et al., 2017, United Kingdom [45]	Prospective Longitudinal	- To investigate the expression of SLAM-family receptors on T lymphocytes, including DNT cells, from SLE patients with different disease activity.	30	M:F = n/a Median 34.5 yrs.	Median (range) 8 yrs. (0–35)	-Active (with nephritis) -Inactive	n = 19 n = 11	n/a n/a	n = 20 M:F = 4:16 Median (range) 34 yrs. (24–54)	CD3+ CD4− CD8−	Mean ± SEM Active: 5.75 ± 3.43 Inactive: 3.68 ± 1.77 Control: 5.25 ± 3.34 I vs. C [p = ns] A vs. C [p = ns] A vs. I [p = ns]	FACSVerse	MMF (n = 16) HCQ (n = 23) AZA (n = 6) Pred (n = 14) None (n = 1)	- The frequency of DNT cells expressing SLAMF2/4/7 receptors was markedly altered in SLE patients, but these differences did not correlate with disease activity. - SLAMF6 expression on DNT cells could correlate with the response to B-cell depletion after rituximab.
Lai et al., 2018, USA [38]	Prospective, Single-arm, Open-label, Phase 1/2 trial	- To assess sirolimus in active SLE patients that were intolerant or resistant to conventional drugs.	40	M:F = 2:38 Mean ± SD (range) 45.4 ± 14.3 yrs. (18–71)	n/a	-	-	-	43 Mean ± SD (range) 45.3 ± 12.7 yrs. Matched for Gender and Ethnicity	CD3+ CD4− CD8−	n/a $	n/a	n/a	- Increased production of IL-4 and-IL-17 by CD4+ T cells and DNT cells at baseline, which was reduced after 12 months of treatment with sirolimus. IFN-γ production increased during sirolimus treatment in both CD4+ and DNT cells. Mean mitochondrial mass in DNT cells was higher in patients than in controls at baseline, and there was a decrease trend during sirolimus treatment.
Alexander et al., 2020, USA [44]	Prospective Cross-sectional	- To investigate the role of DNT cells in SLE and their potential impact on kidney disease.	50	M:F = n/a Range 7–15 yrs.	n/a	-	-	-	Yes n/a	CD3+ CD4− CD8−	Mean ± SD SLE: 10.0 ± 6.1 Controls: 6.5 ± 1.0	LSRII Contessa	n/a	- DNT cells were increased in kidneys of active SLE patients and correlated with kidney function, in terms of BUN levels.
Li et al., 2020, USA [46]	Prospective Cross-sectional	- to study the interaction between marginal-zone macrophages and DNT cells.	n/a	n/a	n/a	-	-	-	Yes n/a	CD3+ CD4− CD8− CD56- TCRαβ+	Done $	n/a	n/a	- DNT cells were significantly increased in blood from SLE patients compared with healthy controls. - Moreover, Ki67+ DNT cells were also more represented in SLE patients (both in blood and kidney biopsies).

Abbreviations: M: male; F: female; n: number; n/a: information not available; yrs.: years; mo: months; SD: standard deviation; SEM: standard error mean; SLE: systemic lupus erythematosus; DNT: double-negative T cells; CD: cluster of differentiation; CD3: cluster of differentiation 3; CD4: cluster of differentiation 4; CD8: cluster of differentiation 8; TCRαβ: T-cell receptor alpha beta; CD56: cluster of differentiation 56; TCRγδ: T-cell receptor gamma delta; IL-4: interleukin-4; IFN-γ: interferon-γ; IL-17: interleukin-17; NAC: N-acetylcysteine; GSH: glutathione; mTOR: mammalian target of rapamycin; SLEDAI-2K: Systemic Lupus Erythematosus Disease Activity Index 2000; SLAMF2/4/7/6: signaling lymphocytic activation molecule family members 2, 4, 7, and 6; BUN: blood urea nitrogen, MMF: mycophenolate mofetil; HCQ: hydroxychloroquine; AZA: azathioprine; Pred: prednisone. ^$: DNT cells were measured, but the DNT cell results are presented only in figure and numerical values are not shown in any table (refer to the last columns of the present table for the main qualitative findings in this regard).