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Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.

Vivinus-Nebot M, Ticchioni M, Mary F, Hofman P, Quaranta V, Rousselle P, Bernard A - J. Cell Biol. (1999)

Bottom Line: In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures.We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect.This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck).

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Sant¿e et de la Recherche M¿edicale, U343, Nice 06202, France.

ABSTRACT
Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

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Laminin 5 but not laminin 2, when added in soluble  form, inhibits human thymocyte proliferation induced by CD3 +  IL-2. (a) CD3 antibodies were first incubated overnight in culture  wells. Next, 10% FCS in RPMI was added and incubated in order  to minimize further protein adsorption on plastic. After washing,  cells (105/well) were added in culture medium containing IL-2 (5  ng/ml) and laminin 5 or laminin 2. Cultures were also performed  in the presence of 10 μg/ml anti-laminin 5 mAb BM165. (b) Effect of soluble laminin 5 on thymocyte proliferation induced by a  mitogenic CD2 mAb pair (GT2 + D66, 10 μg/ml) plus IL-2.
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Figure 4: Laminin 5 but not laminin 2, when added in soluble form, inhibits human thymocyte proliferation induced by CD3 + IL-2. (a) CD3 antibodies were first incubated overnight in culture wells. Next, 10% FCS in RPMI was added and incubated in order to minimize further protein adsorption on plastic. After washing, cells (105/well) were added in culture medium containing IL-2 (5 ng/ml) and laminin 5 or laminin 2. Cultures were also performed in the presence of 10 μg/ml anti-laminin 5 mAb BM165. (b) Effect of soluble laminin 5 on thymocyte proliferation induced by a mitogenic CD2 mAb pair (GT2 + D66, 10 μg/ml) plus IL-2.

Mentions: Since laminins were shown to influence the functional program of numerous cell types, and in particular bulk preparations of laminins were shown to deliver coactivation signals to T cells (10, 58, 66), we investigated the effects of purified laminin 5 on thymocytes. When we coimmobilized laminin 5 on plastic, this induced a weak costimulation with a CD3–TCR signal, as compared with coated fibronectin or laminin 2 (α2β1γ1), another laminin isoform found in the thymus (10; Fig. 3). We then investigated the effect of laminin 5 and laminin 2 presented in soluble form; soluble laminin 5 clearly inhibited the proliferation of thymocytes stimulated via the CD3–TCR (Fig. 4 a). The inhibition was dose-dependent reaching a maximum of 50%, in terms of [3H]thymidine incorporation, at 5 μg/ml. Over six independent experiments we saw a quite significant inhibition down to 1 μg/ml (∼30%). We checked the specificity of laminin 5–induced inhibition by adding the anti-α3 chain mAb BM165, which recognizes an epitope involved in the interaction of laminin 5 with cells. BM165 abolished the inhibiting effect observed with soluble laminin 5 alone (Fig. 4 a). Conversely, no inhibition was seen when we added, instead of laminin 5, increasing amounts of soluble human laminin 2 (Fig. 4 a).


Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.

Vivinus-Nebot M, Ticchioni M, Mary F, Hofman P, Quaranta V, Rousselle P, Bernard A - J. Cell Biol. (1999)

Laminin 5 but not laminin 2, when added in soluble  form, inhibits human thymocyte proliferation induced by CD3 +  IL-2. (a) CD3 antibodies were first incubated overnight in culture  wells. Next, 10% FCS in RPMI was added and incubated in order  to minimize further protein adsorption on plastic. After washing,  cells (105/well) were added in culture medium containing IL-2 (5  ng/ml) and laminin 5 or laminin 2. Cultures were also performed  in the presence of 10 μg/ml anti-laminin 5 mAb BM165. (b) Effect of soluble laminin 5 on thymocyte proliferation induced by a  mitogenic CD2 mAb pair (GT2 + D66, 10 μg/ml) plus IL-2.
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Related In: Results  -  Collection

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Figure 4: Laminin 5 but not laminin 2, when added in soluble form, inhibits human thymocyte proliferation induced by CD3 + IL-2. (a) CD3 antibodies were first incubated overnight in culture wells. Next, 10% FCS in RPMI was added and incubated in order to minimize further protein adsorption on plastic. After washing, cells (105/well) were added in culture medium containing IL-2 (5 ng/ml) and laminin 5 or laminin 2. Cultures were also performed in the presence of 10 μg/ml anti-laminin 5 mAb BM165. (b) Effect of soluble laminin 5 on thymocyte proliferation induced by a mitogenic CD2 mAb pair (GT2 + D66, 10 μg/ml) plus IL-2.
Mentions: Since laminins were shown to influence the functional program of numerous cell types, and in particular bulk preparations of laminins were shown to deliver coactivation signals to T cells (10, 58, 66), we investigated the effects of purified laminin 5 on thymocytes. When we coimmobilized laminin 5 on plastic, this induced a weak costimulation with a CD3–TCR signal, as compared with coated fibronectin or laminin 2 (α2β1γ1), another laminin isoform found in the thymus (10; Fig. 3). We then investigated the effect of laminin 5 and laminin 2 presented in soluble form; soluble laminin 5 clearly inhibited the proliferation of thymocytes stimulated via the CD3–TCR (Fig. 4 a). The inhibition was dose-dependent reaching a maximum of 50%, in terms of [3H]thymidine incorporation, at 5 μg/ml. Over six independent experiments we saw a quite significant inhibition down to 1 μg/ml (∼30%). We checked the specificity of laminin 5–induced inhibition by adding the anti-α3 chain mAb BM165, which recognizes an epitope involved in the interaction of laminin 5 with cells. BM165 abolished the inhibiting effect observed with soluble laminin 5 alone (Fig. 4 a). Conversely, no inhibition was seen when we added, instead of laminin 5, increasing amounts of soluble human laminin 2 (Fig. 4 a).

Bottom Line: In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures.We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect.This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck).

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Sant¿e et de la Recherche M¿edicale, U343, Nice 06202, France.

ABSTRACT
Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

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