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The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits.

Hu JC, Greene CJ, King-Lyons ND, Connell TD - PLoS ONE (2015)

Bottom Line: Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits.However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits.Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, The Witebsky Center for Microbial Pathogenesis and Immunology, The University at Buffalo, Buffalo, New York, United States of America.

ABSTRACT
Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8(+) T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8(+) T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8(+) T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants.

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Enhancement of IL-1 production by WT and chimeric HLT adjuvants.Thioglycollate-induced peritoneal macrophages were treated overnight with LPS and HLT adjuvants in vitro. (A) Concentration of IL-1α in the culture supernatant after overnight treatment. (B) Concentration of IL-1β in the culture supernatant after overnight treatment. (C) Staining of uncleaved pro-IL-1β in peritoneal macrophages after overnight treatment. (D) Expression of pro-IL-1β by peritoneal macrophages after in vitro treatment by WT and chimeric HLT adjuvants. (E) Mean fluorescence intensity (MFI) of CD86 as a marker for activation in treated peritoneal macrophages. Data shown (n = 3). Statistical Analysis: One-way ANOVA with Bonferroni post-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared to all other groups unless indicated.
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pone.0142942.g003: Enhancement of IL-1 production by WT and chimeric HLT adjuvants.Thioglycollate-induced peritoneal macrophages were treated overnight with LPS and HLT adjuvants in vitro. (A) Concentration of IL-1α in the culture supernatant after overnight treatment. (B) Concentration of IL-1β in the culture supernatant after overnight treatment. (C) Staining of uncleaved pro-IL-1β in peritoneal macrophages after overnight treatment. (D) Expression of pro-IL-1β by peritoneal macrophages after in vitro treatment by WT and chimeric HLT adjuvants. (E) Mean fluorescence intensity (MFI) of CD86 as a marker for activation in treated peritoneal macrophages. Data shown (n = 3). Statistical Analysis: One-way ANOVA with Bonferroni post-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared to all other groups unless indicated.

Mentions: LT-IIc is the newest member of the type II HLT family [9, 12, 22, 23]. In comparison to all other type II HLTs, LT-IIc was found to profoundly enhance the production of both IL-1α and IL-1β in LPS-stimulated peritoneal macrophages [23]. To determine if the new chimeric HLT adjuvants, and specifically LT-IIbc, maintained this unique property, the effects of both WT and chimeric HLT on peritoneal macrophages were evaluated. Analogous with the previous report, LT-IIc potentiated significantly more IL-1α and IL-1β secretion in LPS-primed peritoneal macrophages in comparison to LT-IIb and controls (Fig 3A and 3B). Surprisingly, the effects of both LT-IIbc and LT-IIcb on macrophages were similar to those elicited by LT-IIb; neither chimeric HLT elevated secretion of IL-1α to levels equivalent to those elevated by LT-IIc (Fig 3A and 3B). Processing of pro-IL-1β into the mature IL-1β is required prior to release from the cell. Therefore, intracellular levels of pro-IL-1β were examined in these macrophages (Fig 3C). While all HLT treated macrophages had higher levels of intracellular pro-IL-1β than did macrophages treated only with LPS or mock treated controls, levels of intracellular pro-IL-1β were highest in cells treated with LT-IIb (Fig 3C and 3D). Additionally, we found that LT-IIb treated cells also exhibited higher percentages of macrophages that expressed pro-IL-1β (Fig 3C, S1 Fig) when compared to the other groups. This observation was surprising as mature, secreted IL-1β was not significantly elevated in the culture supernatant of LT-IIb-treated macrophages in comparison to the other HLT-treated groups. This result suggested that LT-IIb influences processing and/or expression of pro-IL-1β, thus causing an accumulation of the uncleaved form of the cytokine, a property unique to LT-IIb. Finally, all HLT-treated macrophages exhibited a more activated and mature phenotype when compared to macrophages treated solely with LPS, as measured by the expression of CD86 (Fig 3E). CD86 expression was equivalent among the HLT treated groups.


The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits.

Hu JC, Greene CJ, King-Lyons ND, Connell TD - PLoS ONE (2015)

Enhancement of IL-1 production by WT and chimeric HLT adjuvants.Thioglycollate-induced peritoneal macrophages were treated overnight with LPS and HLT adjuvants in vitro. (A) Concentration of IL-1α in the culture supernatant after overnight treatment. (B) Concentration of IL-1β in the culture supernatant after overnight treatment. (C) Staining of uncleaved pro-IL-1β in peritoneal macrophages after overnight treatment. (D) Expression of pro-IL-1β by peritoneal macrophages after in vitro treatment by WT and chimeric HLT adjuvants. (E) Mean fluorescence intensity (MFI) of CD86 as a marker for activation in treated peritoneal macrophages. Data shown (n = 3). Statistical Analysis: One-way ANOVA with Bonferroni post-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared to all other groups unless indicated.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643920&req=5

pone.0142942.g003: Enhancement of IL-1 production by WT and chimeric HLT adjuvants.Thioglycollate-induced peritoneal macrophages were treated overnight with LPS and HLT adjuvants in vitro. (A) Concentration of IL-1α in the culture supernatant after overnight treatment. (B) Concentration of IL-1β in the culture supernatant after overnight treatment. (C) Staining of uncleaved pro-IL-1β in peritoneal macrophages after overnight treatment. (D) Expression of pro-IL-1β by peritoneal macrophages after in vitro treatment by WT and chimeric HLT adjuvants. (E) Mean fluorescence intensity (MFI) of CD86 as a marker for activation in treated peritoneal macrophages. Data shown (n = 3). Statistical Analysis: One-way ANOVA with Bonferroni post-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 compared to all other groups unless indicated.
Mentions: LT-IIc is the newest member of the type II HLT family [9, 12, 22, 23]. In comparison to all other type II HLTs, LT-IIc was found to profoundly enhance the production of both IL-1α and IL-1β in LPS-stimulated peritoneal macrophages [23]. To determine if the new chimeric HLT adjuvants, and specifically LT-IIbc, maintained this unique property, the effects of both WT and chimeric HLT on peritoneal macrophages were evaluated. Analogous with the previous report, LT-IIc potentiated significantly more IL-1α and IL-1β secretion in LPS-primed peritoneal macrophages in comparison to LT-IIb and controls (Fig 3A and 3B). Surprisingly, the effects of both LT-IIbc and LT-IIcb on macrophages were similar to those elicited by LT-IIb; neither chimeric HLT elevated secretion of IL-1α to levels equivalent to those elevated by LT-IIc (Fig 3A and 3B). Processing of pro-IL-1β into the mature IL-1β is required prior to release from the cell. Therefore, intracellular levels of pro-IL-1β were examined in these macrophages (Fig 3C). While all HLT treated macrophages had higher levels of intracellular pro-IL-1β than did macrophages treated only with LPS or mock treated controls, levels of intracellular pro-IL-1β were highest in cells treated with LT-IIb (Fig 3C and 3D). Additionally, we found that LT-IIb treated cells also exhibited higher percentages of macrophages that expressed pro-IL-1β (Fig 3C, S1 Fig) when compared to the other groups. This observation was surprising as mature, secreted IL-1β was not significantly elevated in the culture supernatant of LT-IIb-treated macrophages in comparison to the other HLT-treated groups. This result suggested that LT-IIb influences processing and/or expression of pro-IL-1β, thus causing an accumulation of the uncleaved form of the cytokine, a property unique to LT-IIb. Finally, all HLT-treated macrophages exhibited a more activated and mature phenotype when compared to macrophages treated solely with LPS, as measured by the expression of CD86 (Fig 3E). CD86 expression was equivalent among the HLT treated groups.

Bottom Line: Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits.However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits.Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, The Witebsky Center for Microbial Pathogenesis and Immunology, The University at Buffalo, Buffalo, New York, United States of America.

ABSTRACT
Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8(+) T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8(+) T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8(+) T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants.

Show MeSH
Related in: MedlinePlus