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Effects of natalizumab treatment on Foxp3+ T regulatory cells.

Stenner MP, Waschbisch A, Buck D, Doerck S, Einsele H, Toyka KV, Wiendl H - PLoS ONE (2008)

Bottom Line: Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions.We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population.We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Julius-Maximilians University, Wuerzburg, Germany.

ABSTRACT

Background: Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology: A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal findings: Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions: We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

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Differential binding of Natalizumab to T cell subsets.(A) Binding of FITC-conjugated Natalizumab (20 µg/ml) to CD4+ and CD8+ T cells derived from patients with a relapsing-remitting disease course (RRMS; n = 9) and (B) to regulatory, Foxp3+ and non-regulatory, Foxp3− CD4+ T cells from healthy donors (HD; n = 9) and RRMS patients (n = 10) as analyzed by flow cytometry. (C) Differences in Natalizumab binding closely reflect surface expression patterns of the α4 chain (CD49d) of VLA-4 as analyzed by flow cytometry. (D) CD49d surface levels were analyzed by flow cytometry before (light grey) and 1 month after initiation of therapy with Natalizumab (dark grey). Postinfusion levels were normalized to the expression of CD49d before treatment. The relative loss of functional VLA-4 is lower in CD4+Foxp3+ T regulatory cells than in conventional CD4+ T cells (n = 15). SFI = specific fluorescent index, * p<0,05; **p<0,005, student's t-test.
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pone-0003319-g001: Differential binding of Natalizumab to T cell subsets.(A) Binding of FITC-conjugated Natalizumab (20 µg/ml) to CD4+ and CD8+ T cells derived from patients with a relapsing-remitting disease course (RRMS; n = 9) and (B) to regulatory, Foxp3+ and non-regulatory, Foxp3− CD4+ T cells from healthy donors (HD; n = 9) and RRMS patients (n = 10) as analyzed by flow cytometry. (C) Differences in Natalizumab binding closely reflect surface expression patterns of the α4 chain (CD49d) of VLA-4 as analyzed by flow cytometry. (D) CD49d surface levels were analyzed by flow cytometry before (light grey) and 1 month after initiation of therapy with Natalizumab (dark grey). Postinfusion levels were normalized to the expression of CD49d before treatment. The relative loss of functional VLA-4 is lower in CD4+Foxp3+ T regulatory cells than in conventional CD4+ T cells (n = 15). SFI = specific fluorescent index, * p<0,05; **p<0,005, student's t-test.

Mentions: We first assessed how Natalizumab binds to Foxp3+ Tregs versus Foxp3 negative CD4+ T cells. After evaluating the saturation of Natalizumab binding sites using FITC-conjugated Natalizumab, a saturating dose of Natalizumab (20 µg/ml) was used to assess a differential binding to T cell subsets. The concentration of 20 µg/ml lies within the range of serum trough levels of treated patients [9]. A comparison of the specific fluorescent indices (SFI) of CD4+ and CD8+ T cells in untreated MS patients revealed significantly higher binding of Natalizumab to CD8+ T cells (Figure 1a). In contrast, binding of Natalizumab to the CD4+Foxp3+ subset of regulatory T cells was significantly lower compared to their CD4+Foxp3 negative counterparts, both in healthy donors and MS patients (Figure 1b). The disease course (acute relapse versus stable; n = 5 each) had no effect on Natalizumab binding to T cell subsets derived from the peripheral blood of MS patients (data not shown).


Effects of natalizumab treatment on Foxp3+ T regulatory cells.

Stenner MP, Waschbisch A, Buck D, Doerck S, Einsele H, Toyka KV, Wiendl H - PLoS ONE (2008)

Differential binding of Natalizumab to T cell subsets.(A) Binding of FITC-conjugated Natalizumab (20 µg/ml) to CD4+ and CD8+ T cells derived from patients with a relapsing-remitting disease course (RRMS; n = 9) and (B) to regulatory, Foxp3+ and non-regulatory, Foxp3− CD4+ T cells from healthy donors (HD; n = 9) and RRMS patients (n = 10) as analyzed by flow cytometry. (C) Differences in Natalizumab binding closely reflect surface expression patterns of the α4 chain (CD49d) of VLA-4 as analyzed by flow cytometry. (D) CD49d surface levels were analyzed by flow cytometry before (light grey) and 1 month after initiation of therapy with Natalizumab (dark grey). Postinfusion levels were normalized to the expression of CD49d before treatment. The relative loss of functional VLA-4 is lower in CD4+Foxp3+ T regulatory cells than in conventional CD4+ T cells (n = 15). SFI = specific fluorescent index, * p<0,05; **p<0,005, student's t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553177&req=5

pone-0003319-g001: Differential binding of Natalizumab to T cell subsets.(A) Binding of FITC-conjugated Natalizumab (20 µg/ml) to CD4+ and CD8+ T cells derived from patients with a relapsing-remitting disease course (RRMS; n = 9) and (B) to regulatory, Foxp3+ and non-regulatory, Foxp3− CD4+ T cells from healthy donors (HD; n = 9) and RRMS patients (n = 10) as analyzed by flow cytometry. (C) Differences in Natalizumab binding closely reflect surface expression patterns of the α4 chain (CD49d) of VLA-4 as analyzed by flow cytometry. (D) CD49d surface levels were analyzed by flow cytometry before (light grey) and 1 month after initiation of therapy with Natalizumab (dark grey). Postinfusion levels were normalized to the expression of CD49d before treatment. The relative loss of functional VLA-4 is lower in CD4+Foxp3+ T regulatory cells than in conventional CD4+ T cells (n = 15). SFI = specific fluorescent index, * p<0,05; **p<0,005, student's t-test.
Mentions: We first assessed how Natalizumab binds to Foxp3+ Tregs versus Foxp3 negative CD4+ T cells. After evaluating the saturation of Natalizumab binding sites using FITC-conjugated Natalizumab, a saturating dose of Natalizumab (20 µg/ml) was used to assess a differential binding to T cell subsets. The concentration of 20 µg/ml lies within the range of serum trough levels of treated patients [9]. A comparison of the specific fluorescent indices (SFI) of CD4+ and CD8+ T cells in untreated MS patients revealed significantly higher binding of Natalizumab to CD8+ T cells (Figure 1a). In contrast, binding of Natalizumab to the CD4+Foxp3+ subset of regulatory T cells was significantly lower compared to their CD4+Foxp3 negative counterparts, both in healthy donors and MS patients (Figure 1b). The disease course (acute relapse versus stable; n = 5 each) had no effect on Natalizumab binding to T cell subsets derived from the peripheral blood of MS patients (data not shown).

Bottom Line: Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions.We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population.We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Julius-Maximilians University, Wuerzburg, Germany.

ABSTRACT

Background: Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology: A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal findings: Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions: We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

Show MeSH
Related in: MedlinePlus