Table 1.

Tolerogenic DCs therapies in EAE and in vitro studies using MS patients tolDCs

Tolerogenic DC therapies in EAEa
Tolerogenic agent	EAE model	Groups of treatment	Number of administrations	Type of treatmentb	Route	Number of tolDCs	Clinical outcome	Mechanism of action	Observations	Ref
K313	C57BL/6 mice immunized with MOG35–55	MOG-K313-DC, unpulsed K313-DC and MOG-VitD3-tolDCs (control group)	3 administrations (every 3 days) at days 10, 13, and 16 p.i.	Early therapeutic	i.v.	1 × 106 cells	Clinical symptoms amelioration (similar to MOG-VitD3-tolDCs group)	Strong reduction of leukocyte infiltration and demyelination Reduction of Th1 and Th17 cells and increase of Treg compared to untreated group	BMDCs treated with K313 inhibit antigen-specific CD4+ T cells	60
VitD3	C57BL/6 mice immunized with MOG35–55	MOG mRNA electroporated-VitD3-tolDCs, MOG-VitD3-tolDCs, unpulsed VitD3-tolDCs and PBS	3 administrations (every 4 days) at days 13, 17, and 21 p.i.	Therapeutic	i.v.	1 × 106 cells	Reduced disease severity with both electroporated and pulsed antigen-specific VitD3-tolDCs	Decrease of number of spinal cord lesions (MRI) Decreased MOG-reactive T cells in the spleen and lymph nodes and reduction of proinflammatory cytokines secretion (IL-17, IFN-γ, TNF-α, GM-CSF)		61
	C57BL/6 mice immunized with MOG35–55	MOG-VitD3-tolDCs, MOG-DC and PBS	3 administrations (every 3 days) at days 10, 13, and 16 p.i.	Early therapeutic	i.v.	0.8 × 106 cells	Delay of disease onset Reduction of EAE severity	Decreased Th1 and Th17 cell infiltration into the CNS Enhanced % of Treg, CD4+ IL10+ T cells and Breg in spleen and/or lymph nodes	No maturated DC	62
	C57BL/6 mice immunized with MOG35–55	MOG and unpulsed engineered tolDCs overespressing VitD3 and untreated EAE	1 single administration at day 10 p.i. or 3 administrations (every 7 days) at days 10, 17, and 24 p.i.	Early therapeutic	i.v. (1 dose) s.c. (3 doses)	1 × 106 cells	Reduced disease severity with antigen-specific manner	Less inflammatory foci and demyelination Increase of Th2, Tr1, and FoxP3+ Treg cells Higher secretion of IL-4 and IL-10 compared to control groups Clinical amelioration related with FoxP3+ Treg induction	DC engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase (self-tolerized) and release 1,25-dihydoxyvitamin D	63
	C57BL/6 mice immunized with MOG40–55	Cryopreserved MOG-VitD3-tolDCs, unpulsed VitD3-tolDCs and PBS	For short-term treatment (30 days), 3 administrations (every 4 days: 15, 19, and 23 p.i.) For long-term treatment (74 days), 3 initial administrations (every 4 days: 14, 18, and 22 p.i.) + extra-administrations when the mean clinical score of the MOG-VitD3-tolDCs group increased	Therapeutic	i.v.	1 × 106 cells	Reduced disease severity following short and long-term antigen-specific treatment with antigen-specific VitD3-tolDCs	Short-term treatment: Inhibition of MOG40–55 T cell reactivity and increased proportion of FoxP3+ Treg Long-term treatment: Inhibition of MOG40–55 T cell reactivity; increase of Breg and activated NKT cells; and decreased proportion and activation of NK cells	The long term-treatment with cryopreserved tolDCs-VitD3-MOG was well tolerated, highly effective and exhibited a prolonged clinical benefit after each administration.	64
	C57BL/6 mice immunized with MOG40–55	MOG-VitD3-tolDCs, unpulsed VitD3-tolDCs and PBS	2 administrations for prophylactic (−2, 5 p.i.) and late-prophylactic (5, 9 p.i.) approaches and 3 administrations (every 4 days: 15, 19, and 23 p.i.) for therapeutic	Prophylactic, late-prophylactic and therapeutic	i.v.	1 × 106 cells	Antigen-specific VitD3-tolDCs showed reduced EAE incidence (prophylactic treatment) and reduced disease severity (late-prophylactic and therapeutic treatment)	Induction of FoxP3+ Treg and IL-10 secretion Inhibition of MOG40-55-specific T cells reactivity	Biodistribution analysis showed that although tolDCs reached CNS following i.v. injection, tolDCs accumulated in spleen after 48 h and remained there at day 14 p.i.	65
Chloroquine	C57BL/6 mice immunized with MOG35–55	MOG-CQ-BMDC, MOG-PBS-BMDC and untreated EAE	1 single administration at day 10 p.i.	Late-prophylactic	i.v.	1.5 × 106 cells	Reduction of EAE severity Suppression of Th17 infiltration into the CNS	Inhibition of T cell proliferation and reduction of differentiation and infiltration of Th17 cells mediated by STAT1 signaling Increase of IL10 secretion and reduction of IFN-γ levels Induction of FoxP3+ Treg	STAT1−/− mice were also used in the experiments and treated with MOG-CQ-BMDC or MOG-PBS-BMDC	66
Tofacitinib	C57BL/6 mice immunized with MOG35–55	MOG-Tofa-DC, unpulsed-Tofa-DC PBS, and non-immunized	3 administrations (every 4 days) at days 7, 11, and 15 p.i.	Early therapeutic	i.v.	1 × 106 cells	Reduction of EAE severity in mice receiving antigen-specific Tofa-DC	Reduced leukocyte infiltration and less extensive demyelination into the CNS Decreased % of Th1 and Th17 cells and enhanced % of CD25 + FoxP3+ Treg in the spleen	Frequencies of Th17 and Th1 cells correlated positively with the clinical score and correlated negatively with the frequency of Treg	67
BD750	C57BL/6 mice immunized with MOG35–55	MOG-BD750-tolDCs, npulsed BD750-tolDCs, PBS and non-immunized	3 administrations (every 4 days) Prophylactic approach: days −2, 2, and 6 p.i. Early therapeutic approach: days 7, 11 and 15 p.i. Late-therapeutic approach: days 19, 23, and 27 p.i.	Prophylactic, early therapeutic and late-therapeutic	i.v.	1 × 106 cells	Early therapeutic treatment: Delay on disease onset and reduced EAE severity with MOG-BD750-tolDCs Late-therapeutic treatment: no clinical improvement	Early therapeutic: Reduced inflammatory infiltrates and demyelination in the CNS Reduced frequency of Th1 and Th17 cells and increased % of FoxP3+ Treg in the spleen	Early therapeutic treatment using only 1 or 2 administrations of MOG-BD750-tolDCs (at day 7 p.i. or days 7 and 11 p.i.) showed no clinical efficacy	68
IL-35	C57BL/6 receiving 5·106 MOG35–55-specific T cells (Passive EAE)	MOG loaded and unpulsed tumor DC cell line modified to express IL-35	1 single administration 1 day after passive EAE induction (prophylactic) and 2 administrations at day 6 and 8 post passive EAE induction (late-prophylactic)	Prophylactic and Late-prophylactic	i.v.	2.5 × 106 cells	Reduction of EAE symptoms	Impairment of T cell activation and proliferation		69
LPS	C57BL/6 mice immunized with MOG35–55	MOG-LPS-DC, MOG-DC and unpulsed DC	3 administrations (every 4 days) at days 11, 14, and 17 p.i.	Early therapeutic	i.v.	0.3 × 106 cells	Antigen-specific abrogation of EAE development	LPS-treated BMDC increase the frequency of Treg (CD4+ CD25+ FoxP3+ GITR+) that are CD127+ 3G11− and inhibit those CD127 + 3G11 + Treg		70
Apoptotic thymocytes	C57BL/6 mice immunized with MOG35–55	Splenic DC primed with irradiated (apoptotic) or fresh T cells and pulsed with or without MOG	3 administrations (every 4 days) at days 11, 14, and 17 p.i.	Early therapeutic	i.v.	0.3 × 106 cells	Antigen-specific abrogation of EAE development	Inhibition of central and effector memory CD4+ T cell development Reduction of IFNγ production	Immune tolerance induced by apoptotic T cell-treated DC is mainly related to CD4+ effector memory T cells reduction	71

In vitro studies using tolDCs from MS patients
Tolerogenic agent	Phenotype		Functionality	Stability	Comparison with HD tolDCs		Mechanism of action		Observations	Ref
VitD3	Reduced expression of CD86, CD83 and HLA-DR molecules Production of higher levels of IL-10 (although not statistically significant) and low levels of IL12p70 compared to mDC		Reduced ability to stimulate T cell allogeneic proliferation Decrease secretion of TNF-α, IL-6, and IFN-γ Induction of antigen-specific T cell hyporesponsiveness	Stable phenotype and functionality LPS stimulation did not induce IL-23 secretion but small amounts of IL-10 were still detectable	Equal efficiency to generate VitD3-tolDCs from MS patients and HD Low level expression of CD83 in mDC from MS patients compared with HD		Not provided		Set up of myelin peptide-tolDCs loading	82
	Decreased expression of CD86, CD80, CD83, and HLA-DR molecules		Reduced allogeneic and antigen-specific T cell proliferation Impaired secretion of proinflammatory cytokines	No tested on fresh VitD3-tolDCs Cryopreserved VitD3-tolDCs remained phenotypically and functionally stables following LPS stimulation	VitD3-tolDCs and mDC from MS patients secreted higher levels of IL-1β, IL-6 and TNF-α compared to HD counterparts		Unknown No induction of neither FoxP3+ Treg nor IL-10 and/or TGF-β producing cells		Cryopreserved VitD3-tolDCs were studied: They retained phenotypical and functional characteristics and remain stable following proinflammatory stimulus	75
	Reduced expression of CD83, CD86, and HLA-DR molecules		Inhibition of allogenic proliferation and increase secretion of IL-10	Non evaluated	VitD3-tolDCs from HD also exhibited increase secretion of TGF-β		No provided		MUCL1 and MAP7 were reported as biomarkers of both HD and MS patients VitD3-tolDCs	87
Ethyl pyruvate (EP)	Decreased expression of CD86, CD83, and HLA-DR molecules		Inhibition of allogenic T cell proliferation with reduction of IFN-γ. No increase of IL-10 levels was observed	Non evaluated	No differences in neither phenotype nor allogenic proliferation inhibition of HD and MS patients		No provided		Similar phenotype and functionality of EP-tolDCs and VitD3-tolDCs	86

CNS central nervous system, CQ chloroquine, DC dendritic cells, EP ethyl pyruvate, HD healthy donors, i.v. intravenous, MAP7 microtubule-associated protein 7, MOG myelin oligodendrocyte glycoprotein, MRI magnetic resonance imaging, MS multiple sclerosis, MUCL1 mucin-like 1, Tofa tofacitinib, VitD3 vitamin D3

^aFrom 2015 to 2020

^bProphylactic treatment: treatment before or at the moment of EAE induction; Late-prophylactic treatment: treatment after EAE induction but prior to clinical onset; early therapeutic treatment: initiation of treatment when mice showed first clinical symptoms; therapeutic treatment: first dose administrated to mice with established clinical signs; late-therapeutic: treatment in mice with high degree of paralysis