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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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Natalizumab blocks both Tregs and non-regulatory T cells in in vitro transmigration assays.CD4+ T cells were analyzed for their migratory capacity in a fibronectin coated Boyden chamber assay system. The relative proportion of Foxp3+ cells before migration (periphery) and within the migrated fraction (migrated) was assessed by multicolour flow cytometry. The bar diagram demonstrates the frequency of Foxp3+ T cells in the two fractions before and after initiation of Natalizumab therapy (n = 15). No differences were observed under any condition.
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pone-0003319-g002: Natalizumab blocks both Tregs and non-regulatory T cells in in vitro transmigration assays.CD4+ T cells were analyzed for their migratory capacity in a fibronectin coated Boyden chamber assay system. The relative proportion of Foxp3+ cells before migration (periphery) and within the migrated fraction (migrated) was assessed by multicolour flow cytometry. The bar diagram demonstrates the frequency of Foxp3+ T cells in the two fractions before and after initiation of Natalizumab therapy (n = 15). No differences were observed under any condition.

Mentions: It is currently unknown how the migratory behaviour differs between Foxp3+ Tregs and Foxp3− CD4+ T cells in MS patients and how blockade of VLA-4 influences migration of Tregs. According to Niino et al., the relative loss of CD49d immunoreactivity under in vivo therapy directly correlates with the migratory behaviour of immune cell subsets as demonstrated for monocytes, B and T lymphocytes [13]. Since expression of CD49d was significantly lower in Foxp3+ Tregs than in Foxp3− T cells and the relative decrease of CD49d immunoreactivity in the presence of Natalizumab was significantly lower on Foxp3+ Tregs compared to conventional CD4+ (Figure 1), we were curious if this finding might be associated with an aberrant migratory behaviour. To assess transmigration of CD4+ T cells we used fibronectin coated Boyden chambers, a well characterized experimental set-up to assess transmigration mediated by interaction of VLA-4 on the T cell surface with its alternative binding partner, the CS-1 fragment of fibronectin [13], [20]. In line with previous findings by Niino et al. Natalizumab blocked T cell transmigration in this experimental setting (data not shown). To find out whether Natalizumab might favour the migration of Tregs, we assessed the percentage of Foxp3+ T cells at baseline and within the migrated fraction. A selective blockade of non-regulatory CD4+ compared to Tregs should have resulted in an enrichment of Foxp3+ T cells within the migrated fraction. However, the percentage of Foxp3+ T cells within the migrated fraction remained the same irrespective of Natalizumab treatment (n = 15, Figure 2).


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)

Natalizumab blocks both Tregs and non-regulatory T cells in in vitro transmigration assays.CD4+ T cells were analyzed for their migratory capacity in a fibronectin coated Boyden chamber assay system. The relative proportion of Foxp3+ cells before migration (periphery) and within the migrated fraction (migrated) was assessed by multicolour flow cytometry. The bar diagram demonstrates the frequency of Foxp3+ T cells in the two fractions before and after initiation of Natalizumab therapy (n = 15). No differences were observed under any condition.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003319-g002: Natalizumab blocks both Tregs and non-regulatory T cells in in vitro transmigration assays.CD4+ T cells were analyzed for their migratory capacity in a fibronectin coated Boyden chamber assay system. The relative proportion of Foxp3+ cells before migration (periphery) and within the migrated fraction (migrated) was assessed by multicolour flow cytometry. The bar diagram demonstrates the frequency of Foxp3+ T cells in the two fractions before and after initiation of Natalizumab therapy (n = 15). No differences were observed under any condition.
Mentions: It is currently unknown how the migratory behaviour differs between Foxp3+ Tregs and Foxp3− CD4+ T cells in MS patients and how blockade of VLA-4 influences migration of Tregs. According to Niino et al., the relative loss of CD49d immunoreactivity under in vivo therapy directly correlates with the migratory behaviour of immune cell subsets as demonstrated for monocytes, B and T lymphocytes [13]. Since expression of CD49d was significantly lower in Foxp3+ Tregs than in Foxp3− T cells and the relative decrease of CD49d immunoreactivity in the presence of Natalizumab was significantly lower on Foxp3+ Tregs compared to conventional CD4+ (Figure 1), we were curious if this finding might be associated with an aberrant migratory behaviour. To assess transmigration of CD4+ T cells we used fibronectin coated Boyden chambers, a well characterized experimental set-up to assess transmigration mediated by interaction of VLA-4 on the T cell surface with its alternative binding partner, the CS-1 fragment of fibronectin [13], [20]. In line with previous findings by Niino et al. Natalizumab blocked T cell transmigration in this experimental setting (data not shown). To find out whether Natalizumab might favour the migration of Tregs, we assessed the percentage of Foxp3+ T cells at baseline and within the migrated fraction. A selective blockade of non-regulatory CD4+ compared to Tregs should have resulted in an enrichment of Foxp3+ T cells within the migrated fraction. However, the percentage of Foxp3+ T cells within the migrated fraction remained the same irrespective of Natalizumab treatment (n = 15, Figure 2).

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