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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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(a) The anti-α6 integrin chain mAb S3-41 has a peculiar  pattern of reactivity with human thymocytes, resembling the reactivity of the anti-β4 chain mAb. Two color immunofluorescence was performed on human thymocytes with the CD3 mAb  X3 directly conjugated with FITC and with the indicated  amounts of anti-integrin mAb that was indirectly labeled with  goat anti–mouse PE conjugated F(ab)′2 fragments. The percentage of positive cells for the integrin chain analyzed is noted in the  upper corner of the corresponding area. α6 expression was analyzed with the mAbs GOH3 and S3-41; β1 expression was analyzed with the mAb K20 and β4 expression was analyzed with the  mAb 3E1F6. (b) Reactivities of the anti-α6 mAb S3-41 and  GOH3 and of the anti-β4 mAb 3E1F6 with thymocyte subpopulations according to their surface density in CD3. The percentage  in the upper right corner indicates the number of positively  stained cells. All antibodies were used at saturating doses, i.e., 10  μg/ml for S3-41, 3E1F6, K20 and 5 μg/ml for GOH3 and X3.
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Figure 6: (a) The anti-α6 integrin chain mAb S3-41 has a peculiar pattern of reactivity with human thymocytes, resembling the reactivity of the anti-β4 chain mAb. Two color immunofluorescence was performed on human thymocytes with the CD3 mAb X3 directly conjugated with FITC and with the indicated amounts of anti-integrin mAb that was indirectly labeled with goat anti–mouse PE conjugated F(ab)′2 fragments. The percentage of positive cells for the integrin chain analyzed is noted in the upper corner of the corresponding area. α6 expression was analyzed with the mAbs GOH3 and S3-41; β1 expression was analyzed with the mAb K20 and β4 expression was analyzed with the mAb 3E1F6. (b) Reactivities of the anti-α6 mAb S3-41 and GOH3 and of the anti-β4 mAb 3E1F6 with thymocyte subpopulations according to their surface density in CD3. The percentage in the upper right corner indicates the number of positively stained cells. All antibodies were used at saturating doses, i.e., 10 μg/ml for S3-41, 3E1F6, K20 and 5 μg/ml for GOH3 and X3.

Mentions: Next, we investigated whether the effect of laminin 5 would be mediated by α6β4, α3β1, or both. We tested a battery of anti-α6 mAbs for their reactivity in flow cytometry with dispersed human thymocytes (135 13C [rat IgG2a]; J8H, BQ16, 450 30A1, S3-41 [mouse IgG1]; reference 24) and the widely used anti-α6 GOH3 [rat IgG1]; reference 61). We noticed that the mouse mAb S3-41 displayed a peculiar reactivity as compared with other anti-α6 mAbs, strikingly similar to the reactivity of the anti-β4 mAb 3E1F6 (Fig. 6). S3-41, as 3E1F6, did not react with the most immature CD3low thymocytes. Moreover, staining by S3-41 and 3E1F6 increased with the density of CD3, which indicates the level of maturation of thymocytes (Fig. 6). This pattern of reactivity was in marked contrast to the pattern given by the other anti-α6 mAbs tested including GOH3, which stained immature CD3low thymocytes. Of note, peripheral blood T lymphocytes were equally stained with GOH3 and S3-41 (not shown).


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)

(a) The anti-α6 integrin chain mAb S3-41 has a peculiar  pattern of reactivity with human thymocytes, resembling the reactivity of the anti-β4 chain mAb. Two color immunofluorescence was performed on human thymocytes with the CD3 mAb  X3 directly conjugated with FITC and with the indicated  amounts of anti-integrin mAb that was indirectly labeled with  goat anti–mouse PE conjugated F(ab)′2 fragments. The percentage of positive cells for the integrin chain analyzed is noted in the  upper corner of the corresponding area. α6 expression was analyzed with the mAbs GOH3 and S3-41; β1 expression was analyzed with the mAb K20 and β4 expression was analyzed with the  mAb 3E1F6. (b) Reactivities of the anti-α6 mAb S3-41 and  GOH3 and of the anti-β4 mAb 3E1F6 with thymocyte subpopulations according to their surface density in CD3. The percentage  in the upper right corner indicates the number of positively  stained cells. All antibodies were used at saturating doses, i.e., 10  μg/ml for S3-41, 3E1F6, K20 and 5 μg/ml for GOH3 and X3.
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Figure 6: (a) The anti-α6 integrin chain mAb S3-41 has a peculiar pattern of reactivity with human thymocytes, resembling the reactivity of the anti-β4 chain mAb. Two color immunofluorescence was performed on human thymocytes with the CD3 mAb X3 directly conjugated with FITC and with the indicated amounts of anti-integrin mAb that was indirectly labeled with goat anti–mouse PE conjugated F(ab)′2 fragments. The percentage of positive cells for the integrin chain analyzed is noted in the upper corner of the corresponding area. α6 expression was analyzed with the mAbs GOH3 and S3-41; β1 expression was analyzed with the mAb K20 and β4 expression was analyzed with the mAb 3E1F6. (b) Reactivities of the anti-α6 mAb S3-41 and GOH3 and of the anti-β4 mAb 3E1F6 with thymocyte subpopulations according to their surface density in CD3. The percentage in the upper right corner indicates the number of positively stained cells. All antibodies were used at saturating doses, i.e., 10 μg/ml for S3-41, 3E1F6, K20 and 5 μg/ml for GOH3 and X3.
Mentions: Next, we investigated whether the effect of laminin 5 would be mediated by α6β4, α3β1, or both. We tested a battery of anti-α6 mAbs for their reactivity in flow cytometry with dispersed human thymocytes (135 13C [rat IgG2a]; J8H, BQ16, 450 30A1, S3-41 [mouse IgG1]; reference 24) and the widely used anti-α6 GOH3 [rat IgG1]; reference 61). We noticed that the mouse mAb S3-41 displayed a peculiar reactivity as compared with other anti-α6 mAbs, strikingly similar to the reactivity of the anti-β4 mAb 3E1F6 (Fig. 6). S3-41, as 3E1F6, did not react with the most immature CD3low thymocytes. Moreover, staining by S3-41 and 3E1F6 increased with the density of CD3, which indicates the level of maturation of thymocytes (Fig. 6). This pattern of reactivity was in marked contrast to the pattern given by the other anti-α6 mAbs tested including GOH3, which stained immature CD3low thymocytes. Of note, peripheral blood T lymphocytes were equally stained with GOH3 and S3-41 (not shown).

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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