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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 plasma cell generation and cytokine microenvironment. B lymphocytes were expanded for 19 days with IL-2, IL-4, and IL-10 at high interaction level with CD154. The same interleukin combination was used for the differentiation phase (filled symbols and bars) or the cells were cultured from day 19 with a combination of IL-6 and IL-10 (empty symbols and bars). The transition and differentiation phases lasted a total of 18 days with a low CD154 interaction. The results shown are the mean of 6 independent experiments. (a) Cell expansion, (b) cell viability, (c) IgG secretion rate in the differentiation environment, from D19 to D38, (d) IgG, A, and M secretion on day 33 supernatants, and (e) IgG subclasses secretion on day 33 supernatants. A significant difference was noticed in IgG1 secretion among the two interleukins combinations. Statistical analyses were done using the Bonferroni t-test and the P value was <0.05 (f) CD38 and CD38+CD138+ cells frequency. The IL-6-10 combination generated a larger CD38+ cell population on day 33, confirmed by a paired t-test, P value = 0.0188. All errors bars stand for SD and can be smaller than symbols.
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fig1: In vitro plasma cell generation and cytokine microenvironment. B lymphocytes were expanded for 19 days with IL-2, IL-4, and IL-10 at high interaction level with CD154. The same interleukin combination was used for the differentiation phase (filled symbols and bars) or the cells were cultured from day 19 with a combination of IL-6 and IL-10 (empty symbols and bars). The transition and differentiation phases lasted a total of 18 days with a low CD154 interaction. The results shown are the mean of 6 independent experiments. (a) Cell expansion, (b) cell viability, (c) IgG secretion rate in the differentiation environment, from D19 to D38, (d) IgG, A, and M secretion on day 33 supernatants, and (e) IgG subclasses secretion on day 33 supernatants. A significant difference was noticed in IgG1 secretion among the two interleukins combinations. Statistical analyses were done using the Bonferroni t-test and the P value was <0.05 (f) CD38 and CD38+CD138+ cells frequency. The IL-6-10 combination generated a larger CD38+ cell population on day 33, confirmed by a paired t-test, P value = 0.0188. All errors bars stand for SD and can be smaller than symbols.

Mentions: The combination of IL-2, IL-4, and IL-10 along with high CD40-CD154 interaction has been proven to stimulate long-term memory B cell expansion in vitro [42]. Based on the fact that a lower CD40-CD154 interaction leads to B cell differentiation [15], we have compared the IL-2, IL-4, and IL-10 cytokine combination to that of IL-6 and IL-10 in promoting in vitro differentiation at a low CD40-CD154 interaction. This comparison was based on the capacity of either combination to induce immunoglobulin secretion and emergence of CD38+ or CD38hiCD138+ cells. Cells were expanded for 19 days in a medium containing IL-2, IL-4, and IL-10 [16, 42] followed by a 4-day-long transition phase and a 18-day differentiation phase with IL-2-4-10 or IL-6-10 combinations. As shown in Figure 1(a), the passage to the differentiation environment in both conditions slowed cell expansion (Figure 1(a)) and allowed to maintain cell viability above 80% (Figure 1(b)). Being an important feature of B cell differentiation, immunoglobulins secretion was measured in both conditions. IgG secretion rate reached up to 11 ± 2 μg/106 cells/h with the IL-2-4-10 combination and 13 ± 2 μg/106 cells/h with the IL-6-10 combination on day 33 (Figure 1(c)). Differentiated cells also showed an important IgA secretion in supernatants, reaching 43 ± 12 μg/mL for the IL-2-4-10 combination and 48 ± 19 μg/mL for the IL-6-10 combination, while having as expected a negligible IgM secretion in both conditions (Figure 1(d)). On day 38, cells cultured with the IL-6-10 combination had an IgG1 secretion of 203 ± 157 μg/mL, which was almost twofold higher than the 106 ± 65 μg/mL measured for the IL-2-4-10 condition (Bonferroni t-test P < 0.05) (Figure 1(e)). As for phenotypic cell differentiation, we noticed a more important CD38+ cell population with the IL-6-10 combination, reaching up to 51 ± 7% of all CD45+CD19+ cells by day 33. Both interleukins combinations led to the emergence of CD38+CD138+ plasma cells, 23 ± 4% for IL-2-4-10, and 38 ± 4% for IL-6-10 (Figure 1(f)). Overall, the IL-6-10 combination enhanced IgG1 secretion and generated a higher frequency of CD38+ cells indicating that this cytokine combination was more favorable to the in vitro generation of 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 plasma cell generation and cytokine microenvironment. B lymphocytes were expanded for 19 days with IL-2, IL-4, and IL-10 at high interaction level with CD154. The same interleukin combination was used for the differentiation phase (filled symbols and bars) or the cells were cultured from day 19 with a combination of IL-6 and IL-10 (empty symbols and bars). The transition and differentiation phases lasted a total of 18 days with a low CD154 interaction. The results shown are the mean of 6 independent experiments. (a) Cell expansion, (b) cell viability, (c) IgG secretion rate in the differentiation environment, from D19 to D38, (d) IgG, A, and M secretion on day 33 supernatants, and (e) IgG subclasses secretion on day 33 supernatants. A significant difference was noticed in IgG1 secretion among the two interleukins combinations. Statistical analyses were done using the Bonferroni t-test and the P value was <0.05 (f) CD38 and CD38+CD138+ cells frequency. The IL-6-10 combination generated a larger CD38+ cell population on day 33, confirmed by a paired t-test, P value = 0.0188. All errors bars stand for SD and can be smaller than symbols.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: In vitro plasma cell generation and cytokine microenvironment. B lymphocytes were expanded for 19 days with IL-2, IL-4, and IL-10 at high interaction level with CD154. The same interleukin combination was used for the differentiation phase (filled symbols and bars) or the cells were cultured from day 19 with a combination of IL-6 and IL-10 (empty symbols and bars). The transition and differentiation phases lasted a total of 18 days with a low CD154 interaction. The results shown are the mean of 6 independent experiments. (a) Cell expansion, (b) cell viability, (c) IgG secretion rate in the differentiation environment, from D19 to D38, (d) IgG, A, and M secretion on day 33 supernatants, and (e) IgG subclasses secretion on day 33 supernatants. A significant difference was noticed in IgG1 secretion among the two interleukins combinations. Statistical analyses were done using the Bonferroni t-test and the P value was <0.05 (f) CD38 and CD38+CD138+ cells frequency. The IL-6-10 combination generated a larger CD38+ cell population on day 33, confirmed by a paired t-test, P value = 0.0188. All errors bars stand for SD and can be smaller than symbols.
Mentions: The combination of IL-2, IL-4, and IL-10 along with high CD40-CD154 interaction has been proven to stimulate long-term memory B cell expansion in vitro [42]. Based on the fact that a lower CD40-CD154 interaction leads to B cell differentiation [15], we have compared the IL-2, IL-4, and IL-10 cytokine combination to that of IL-6 and IL-10 in promoting in vitro differentiation at a low CD40-CD154 interaction. This comparison was based on the capacity of either combination to induce immunoglobulin secretion and emergence of CD38+ or CD38hiCD138+ cells. Cells were expanded for 19 days in a medium containing IL-2, IL-4, and IL-10 [16, 42] followed by a 4-day-long transition phase and a 18-day differentiation phase with IL-2-4-10 or IL-6-10 combinations. As shown in Figure 1(a), the passage to the differentiation environment in both conditions slowed cell expansion (Figure 1(a)) and allowed to maintain cell viability above 80% (Figure 1(b)). Being an important feature of B cell differentiation, immunoglobulins secretion was measured in both conditions. IgG secretion rate reached up to 11 ± 2 μg/106 cells/h with the IL-2-4-10 combination and 13 ± 2 μg/106 cells/h with the IL-6-10 combination on day 33 (Figure 1(c)). Differentiated cells also showed an important IgA secretion in supernatants, reaching 43 ± 12 μg/mL for the IL-2-4-10 combination and 48 ± 19 μg/mL for the IL-6-10 combination, while having as expected a negligible IgM secretion in both conditions (Figure 1(d)). On day 38, cells cultured with the IL-6-10 combination had an IgG1 secretion of 203 ± 157 μg/mL, which was almost twofold higher than the 106 ± 65 μg/mL measured for the IL-2-4-10 condition (Bonferroni t-test P < 0.05) (Figure 1(e)). As for phenotypic cell differentiation, we noticed a more important CD38+ cell population with the IL-6-10 combination, reaching up to 51 ± 7% of all CD45+CD19+ cells by day 33. Both interleukins combinations led to the emergence of CD38+CD138+ plasma cells, 23 ± 4% for IL-2-4-10, and 38 ± 4% for IL-6-10 (Figure 1(f)). Overall, the IL-6-10 combination enhanced IgG1 secretion and generated a higher frequency of CD38+ cells indicating that this cytokine combination was more favorable to the in vitro generation of 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