Myelin Oligodendrocyte Glycoprotein (MOG)35-55 Mannan Conjugate Induces Human T-Cell Tolerance and Can Be Used as a Personalized Therapy for Multiple Sclerosis

Abstract

We have previously performed preclinical studies with the oxidized mannan-conjugated peptide MOG35-55 (OM-MOG35-55) in vivo (EAE mouse model) and in vitro (human peripheral blood) and demonstrated that OM-MOG35-55 suppresses antigen-specific T cell responses associated with autoimmune demyelination. Based on these results, we developed different types of dendritic cells (DCs) from the peripheral blood monocytes of patients with multiple sclerosis (MS) or healthy controls presenting OM-MOG35-55 or MOG-35-55 to autologous T cells to investigate the tolerogenic potential of OM-MOG35-55 for its possible use in MS therapy. To this end, monocytes were differentiated into different DC types in the presence of IL-4+GM-CSF ± dexamethasone (DEXA) ± vitamin D3 (VITD3). At the end of their differentiation, the DCs were loaded with peptides and co-cultured with T cells +IL-2 for 4 antigen presentation cycles. The phenotypes of the DC and T cell populations were analyzed using flow cytometry and the secreted cytokines using flow cytometry or ELISA. On day 8, the monocytes had converted into DCs expressing the typical markers of mature or immature phenotypes. Co-culture of T cells with all DC types for 4 antigen presentation cycles resulted in an increase in memory CD4+ T cells compared to memory CD8+ T cells and a suppressive shift in secreted cytokines, mainly due to increased TGF-β1 levels. The best tolerogenic effect was obtained when patient CD4+ T cells were co-cultured with VITD3-DCs presenting OM-MOG35-55, resulting in the highest levels of CD4+PD-1+ T cells and CD4+CD25+Foxp3+ Τ cells. In conclusion, the tolerance induction protocols presented in this work demonstrate that OM-MOG35-55 could form the basis for the development of personalized therapeutic vaccines or immunomodulatory treatments for MS.

Keywords: MOG35–55; cytokines; dendritic cells; human; immunomodulation; mannan; peptides; regulatory T cells; vitamin D.

Figure 1

Protocol for in vitro differentiation of peripheral blood monocytes into different types of DCs presenting peptides to T cells. PEP, peptide; LCs, lymphocytes. This image was created with BioRender (https://biorender.com, accessed on 28 November 2023).

Figure 2

Phenotypic markers on the surface of cultured monocytes on day 0 and DCs on day 8. The results shown are from patient-derived cells. Data are presented as mean (SD). Asterisks indicate statistically significant differences between markers expressed on monocytes on day 0 and DCs on day 8 (* p < 0.05). MFI, mean fluorescence intensity; CTRL, control DCs; DEXA, DEXA-DCs; VITD3, VITD3-DCs; V+D, VITD3+DEXA; d0, day 0.

Figure 3

Cytokines secreted by DCs generated from controls or RRMS patients under different culture conditions on day 8 of culture. Data are presented as mean (SD). Asterisks indicate statistically significant differences between cytokine levels secreted by patient- and control-derived DCs (* p < 0.05).

Figure 4

Ratio of type-2/type-1 cytokines in DC cultures on day 8. Data are presented as mean (SD). Asterisks indicate statistically significant differences between the ratios of type-2/type-1 cytokines (* p < 0.05). Type-2/type-1: [IL-10+TGF-β]:[IL-1+IL-6+IL-8+IL-12+TNF-α].

Figure 5

Phenotypic analysis of (A) lymphocytes, (B) naive and memory CD4+ T cells and (C) naive and memory CD8+ T cells added to DC cultures on day 8. Data are presented as mean (SD). Asterisks indicate statistically significant differences between cell concentrations in patients and controls (* p < 0.05). CD45RA+, naive cells; CD45RO+, memory cells.

Figure 6

Phenotypic analysis of (A) CD4+ T cells, (B) CD8+ T cells and (C) CD4-CD8-T cells on days 33 and 36 of culture with DCs presenting the peptide OM-MOG35–55 (PEP) or the peptide MOG-35–55 (PEPc). Data are presented as mean (SD). Asterisks indicate statistically significant differences between cell levels (* p < 0.05, ** p < 0.01).

Figure 7

Phenotypic analysis of (A) naive CD4+ T cells, (B) memory CD4+ T cells, (C) naive CD8+ T cells and (D) memory CD8+ T cells on days 33 and 36 of culture with DCs presenting the peptide OM-MOG35–55 (PEP) or the peptide MOG-35–55 (PEPc). Data are presented as mean (SD). Asterisks indicate statistically significant differences between cell levels (* p < 0.05, ** p < 0.01). (%), data are presented as % of total CD4+ or CD8+ T cell populations.

Figure 8

Percentage of (A) CD4+PD-1+ T cells and (B) CD4+CD25+Foxp3+ Tregs on days 33 and 36 of culture with DCs presenting peptide OM-MOG35–55 (PEP) or peptide MOG-35–55 (PEPc). Asterisks indicate statistically significant differences between CD4+PD-1+ T cell or Treg levels in cultures with control-derived T cells (gray bars) and patient-derived T cells (black bars). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 9

Cytokine concentration in supernatants of 36-day cultures of DCs and T cells derived from RRMS patients and controls. Cytokine concentrations are shown as mean (SD). Type-2/type-1 cytokine ratio: [IL-4+IL-10+TGF-β1]: [IFN-γ+IL-6+TNF-α]. * p < 0.05, ** p < 0.01. PEP, OM-MOG35–55.

Figure 10

Cytokine concentration in supernatants of 36-day cultures of DCs and T cells derived from RRMS patients and controls. Cytokine concentrations are shown as mean (SD). Type-2/type-1 cytokine ratio: [IL-10+TGF-β1]: [IFN-γ+IL-6+TNF-α]. * p < 0.05, ** p < 0.01. PEPc, MOG35–55.