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Human CD38hiCD138⁺ plasma cells can be generated in vitro from CD40-activated switched-memory B lymphocytes.

Maïga RI, Bonnaure G, Rochette JT, Néron S - J Immunol Res (2014)

Bottom Line: Furthermore, the generated CD38(hi)CD138(+) cells showed a higher proportion of CD31(+) cells than the CD38(hi)CD138(-) cells.Besides, analyses done with human blood and bone marrow plasma cells showed that in vivo and de novo generated CD38(hi)CD138(+) cells have a similar CD31 expression profile but are distinct according to their reduced CD39 expression level.Overall, we have evidences that in vitro generated plasma cells are heterogeneous and appear as CD39(+) precursors to the ones present in bone marrow niches.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Bio-Informatics, Laval University, 1045 Avenue de la Médecine, Québec, QC, Canada G1V 0A6 ; Hema-Quebec's Department of Research and Development, 1070 Avenue des Sciences-de-la-Vie, Québec, QC, Canada G1V 5C3.

ABSTRACT
B lymphocyte differentiation into long-lived plasma cells is the keystone event for the production of long-term protective antibodies. CD40-CD154 and CD27-CD70 interactions are involved in human B lymphocyte differentiation into CD38(hi)CD138(+) cells in vivo as well as in vitro. In this study, we have compared these interactions in their capacity to drive switched-memory B lymphocytes differentiation into CD38(hi)CD138(+) plasma cells. The targeted B lymphocytes were isolated from human peripheral blood, expanded for 19 days, and then submitted to CD70 or CD154 interactions for 14 days. The expanded B lymphocytes were constitutively expressing CD39, whereas CD31's expression was noticed only following the in vitro differentiation step (day 5) and was exclusively present on the CD38(hi) cell population. Furthermore, the generated CD38(hi)CD138(+) cells showed a higher proportion of CD31(+) cells than the CD38(hi)CD138(-) cells. Besides, analyses done with human blood and bone marrow plasma cells showed that in vivo and de novo generated CD38(hi)CD138(+) cells have a similar CD31 expression profile but are distinct according to their reduced CD39 expression level. Overall, we have evidences that in vitro generated plasma cells are heterogeneous and appear as CD39(+) precursors to the ones present in bone marrow niches.

Show MeSH
In vitro generated plasma cells secrete high level of IgA and IgG. Immunoglobulin secretion in supernatants was measured by the Bio-Plex human isotyping kit. The cumulated Ig concentration was determined on (a) day 5 (D5) after the transition phase and (b) day 14 (D14) at the end of differentiation. (c) Relative secretion was determined on D14 by normalizing immunoglobulin concentrations with total cell count on day 5, corresponding to the plateau in proliferation. The results are presented as the mean ± S.E.M. of 6 independent samples. No differences were observed between CD70 and CD154 conditions.
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fig4: In vitro generated plasma cells secrete high level of IgA and IgG. Immunoglobulin secretion in supernatants was measured by the Bio-Plex human isotyping kit. The cumulated Ig concentration was determined on (a) day 5 (D5) after the transition phase and (b) day 14 (D14) at the end of differentiation. (c) Relative secretion was determined on D14 by normalizing immunoglobulin concentrations with total cell count on day 5, corresponding to the plateau in proliferation. The results are presented as the mean ± S.E.M. of 6 independent samples. No differences were observed between CD70 and CD154 conditions.

Mentions: To further compare the B cells generated by each interaction, immunoglobulins concentration was determined in culture supernatants on days 5 (Figure 4(a)) and 14 (Figure 4(b)). As shown, five days into the differentiation environment led to equivalent IgA and IgG secretion from cells in both interaction settings. IgG1 was as expected, according to human serum level, the most secreted IgG subclass with a concentration of 51 ± 10 μg/mL following CD70 interaction and 59 ± 17 μg/mL following CD154 interaction. IgA was secreted at a very high level, with 244 ± 137 μg/mL following CD70 interaction and 211 ± 112 μg/mL following CD154 interaction. However in contrast to IgG1, IgA secretion was highly variable among samples, ranging from 36 to 915 μg/mL for CD70 interaction on day 5. IgG1 concentration was maintained for cells cultured with CD154 until day 14 (51 ± 10 μg/mL), while cells cultured with CD70 showed a decreased secretion of that immunoglobulin subclass (29 ± 4 μg/mL). Moreover, IgG3 secretion with CD154 interaction (17 ± 4 μg/mL) was also twofold higher than with the CD70 interaction (7 ± 2 μg/mL). Based on the high viability and absence of proliferation during the transition phase (D0 to D5), a normalization according to cell concentrations on day 5 was done in order to compare secretion levels on day 14 (Figure 4(c)). Overall, all immunoglobulin secretion rates were equivalent in both conditions indicating similar differentiation status of generated plasmablasts and/or plasma cells.


Human CD38hiCD138⁺ plasma cells can be generated in vitro from CD40-activated switched-memory B lymphocytes.

Maïga RI, Bonnaure G, Rochette JT, Néron S - J Immunol Res (2014)

In vitro generated plasma cells secrete high level of IgA and IgG. Immunoglobulin secretion in supernatants was measured by the Bio-Plex human isotyping kit. The cumulated Ig concentration was determined on (a) day 5 (D5) after the transition phase and (b) day 14 (D14) at the end of differentiation. (c) Relative secretion was determined on D14 by normalizing immunoglobulin concentrations with total cell count on day 5, corresponding to the plateau in proliferation. The results are presented as the mean ± S.E.M. of 6 independent samples. No differences were observed between CD70 and CD154 conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352507&req=5

fig4: In vitro generated plasma cells secrete high level of IgA and IgG. Immunoglobulin secretion in supernatants was measured by the Bio-Plex human isotyping kit. The cumulated Ig concentration was determined on (a) day 5 (D5) after the transition phase and (b) day 14 (D14) at the end of differentiation. (c) Relative secretion was determined on D14 by normalizing immunoglobulin concentrations with total cell count on day 5, corresponding to the plateau in proliferation. The results are presented as the mean ± S.E.M. of 6 independent samples. No differences were observed between CD70 and CD154 conditions.
Mentions: To further compare the B cells generated by each interaction, immunoglobulins concentration was determined in culture supernatants on days 5 (Figure 4(a)) and 14 (Figure 4(b)). As shown, five days into the differentiation environment led to equivalent IgA and IgG secretion from cells in both interaction settings. IgG1 was as expected, according to human serum level, the most secreted IgG subclass with a concentration of 51 ± 10 μg/mL following CD70 interaction and 59 ± 17 μg/mL following CD154 interaction. IgA was secreted at a very high level, with 244 ± 137 μg/mL following CD70 interaction and 211 ± 112 μg/mL following CD154 interaction. However in contrast to IgG1, IgA secretion was highly variable among samples, ranging from 36 to 915 μg/mL for CD70 interaction on day 5. IgG1 concentration was maintained for cells cultured with CD154 until day 14 (51 ± 10 μg/mL), while cells cultured with CD70 showed a decreased secretion of that immunoglobulin subclass (29 ± 4 μg/mL). Moreover, IgG3 secretion with CD154 interaction (17 ± 4 μg/mL) was also twofold higher than with the CD70 interaction (7 ± 2 μg/mL). Based on the high viability and absence of proliferation during the transition phase (D0 to D5), a normalization according to cell concentrations on day 5 was done in order to compare secretion levels on day 14 (Figure 4(c)). Overall, all immunoglobulin secretion rates were equivalent in both conditions indicating similar differentiation status of generated plasmablasts and/or plasma cells.

Bottom Line: Furthermore, the generated CD38(hi)CD138(+) cells showed a higher proportion of CD31(+) cells than the CD38(hi)CD138(-) cells.Besides, analyses done with human blood and bone marrow plasma cells showed that in vivo and de novo generated CD38(hi)CD138(+) cells have a similar CD31 expression profile but are distinct according to their reduced CD39 expression level.Overall, we have evidences that in vitro generated plasma cells are heterogeneous and appear as CD39(+) precursors to the ones present in bone marrow niches.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Bio-Informatics, Laval University, 1045 Avenue de la Médecine, Québec, QC, Canada G1V 0A6 ; Hema-Quebec's Department of Research and Development, 1070 Avenue des Sciences-de-la-Vie, Québec, QC, Canada G1V 5C3.

ABSTRACT
B lymphocyte differentiation into long-lived plasma cells is the keystone event for the production of long-term protective antibodies. CD40-CD154 and CD27-CD70 interactions are involved in human B lymphocyte differentiation into CD38(hi)CD138(+) cells in vivo as well as in vitro. In this study, we have compared these interactions in their capacity to drive switched-memory B lymphocytes differentiation into CD38(hi)CD138(+) plasma cells. The targeted B lymphocytes were isolated from human peripheral blood, expanded for 19 days, and then submitted to CD70 or CD154 interactions for 14 days. The expanded B lymphocytes were constitutively expressing CD39, whereas CD31's expression was noticed only following the in vitro differentiation step (day 5) and was exclusively present on the CD38(hi) cell population. Furthermore, the generated CD38(hi)CD138(+) cells showed a higher proportion of CD31(+) cells than the CD38(hi)CD138(-) cells. Besides, analyses done with human blood and bone marrow plasma cells showed that in vivo and de novo generated CD38(hi)CD138(+) cells have a similar CD31 expression profile but are distinct according to their reduced CD39 expression level. Overall, we have evidences that in vitro generated plasma cells are heterogeneous and appear as CD39(+) precursors to the ones present in bone marrow niches.

Show MeSH