Limits...
Immunomodulation of human intestinal T cells by the synthetic CD80 antagonist RhuDex®.

Heninger AK, Wentrup S, Al-Saeedi M, Schiessling S, Giese T, Wartha F, Meuer S, Schröder-Braunstein J - Immun Inflamm Dis (2014)

Bottom Line: Results show that RhuDex® caused a profound reduction of LPL and PBL proliferation, while Abatacept (CTLA-4-Ig) inhibited LPL proliferation to a small degree, and had no effect on PBL proliferation.Furthermore, Abatacept significantly inhibited IL-2, TNF-α, and IFN-γ release from LPL, primarily produced by CD4(+) T cells, where IL-2 blockage was surprisingly strong, suggesting a down-regulating effect on regulatory T cells.Thus, RhuDex® efficiently inhibited lamina propria and peripheral blood T-cell activation in this pre-clinical study making it a promising drug candidate for the treatment of intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunology, University Hospital Heidelberg Im Neuenheimer Feld 305, 69120, Heidelberg, Germany.

ABSTRACT
Deregulated activation of mucosal lamina propria T cells plays a central role in the pathogenesis of intestinal inflammation. One of the means to attenuate T cell activation is by blocking the CD28/CD80 co-stimulatory pathway. Here we investigate RhuDex®, a small molecule that binds to human CD80, for its effects on the activation of lamina propria T cells employing a gut-culture model of inflammation. To this end, lamina propria leukocytes (LPL) and peripheral blood lymphocytes (PBL) were stimulated either through the CD3/T-cell-receptor complex or the CD2-receptor (CD2) employing agonistic monoclonal antibodies. Co-stimulatory signals were provided by CD80/CD86 present on lamina propria myeloid cells or LPS-activated peripheral blood monocytes. Results show that RhuDex® caused a profound reduction of LPL and PBL proliferation, while Abatacept (CTLA-4-Ig) inhibited LPL proliferation to a small degree, and had no effect on PBL proliferation. Furthermore, Abatacept significantly inhibited IL-2, TNF-α, and IFN-γ release from LPL, primarily produced by CD4(+) T cells, where IL-2 blockage was surprisingly strong, suggesting a down-regulating effect on regulatory T cells. In contrast, in the presence of RhuDex®, secretion of IL-17, again mostly by CD4(+) T cells, and IFN-γ was inhibited in LPL and PBL, yet IL-2 remained unaffected. Thus, RhuDex® efficiently inhibited lamina propria and peripheral blood T-cell activation in this pre-clinical study making it a promising drug candidate for the treatment of intestinal inflammation.

No MeSH data available.


Related in: MedlinePlus

Expression of CD80 and CD86 on WO-LPL and PBMO. (A) Representative FACS plots of WO-LPL harvested after 36 h of organ culture and stained for surface expression of CD33 and CD14 (upper panel). Further, the surface expression of CD80 and CD86 of CD33+ WO-LPMO (lower panel) is shown. Numbers in each quadrant indicate %. (B) Peripheral blood monocytes (PBMO) were isolated from autologous PB using magnetic beads and activated with 1 µg/mL LPS for 8 h to induce CD80 expression. Representative FACS plots showing the purity of isolated CD14+CD33+ PBMO (upper panel) and their expression of CD80 in the absence or presence of LPS activation (lower panel). (C) CD80 (left panel) and CD86 (right panel) surface expression (%) of CD33+ WO-LPMO (7 tissue donors) and CD14+CD33+ PBMO (autologous: PB from 4 of the tissue donors; PB from 4 allogeneic donors).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257762&req=5

fig01: Expression of CD80 and CD86 on WO-LPL and PBMO. (A) Representative FACS plots of WO-LPL harvested after 36 h of organ culture and stained for surface expression of CD33 and CD14 (upper panel). Further, the surface expression of CD80 and CD86 of CD33+ WO-LPMO (lower panel) is shown. Numbers in each quadrant indicate %. (B) Peripheral blood monocytes (PBMO) were isolated from autologous PB using magnetic beads and activated with 1 µg/mL LPS for 8 h to induce CD80 expression. Representative FACS plots showing the purity of isolated CD14+CD33+ PBMO (upper panel) and their expression of CD80 in the absence or presence of LPS activation (lower panel). (C) CD80 (left panel) and CD86 (right panel) surface expression (%) of CD33+ WO-LPMO (7 tissue donors) and CD14+CD33+ PBMO (autologous: PB from 4 of the tissue donors; PB from 4 allogeneic donors).

Mentions: Because RhuDex® binds to CD80, we ensured the presence of CD80 on immunocompetent cells emigrating from our gut-culture model of general inflammation, following EDTA-mediated loss of the epithelial layer. As shown in Fig. 1(A, C) “Walk-Out” lamina propria myeloid cells (CD66b−CD33+ WO-LPMO) express high amounts of CD80 and CD86 (% CD80+: 91.3 ± 3.5; % CD86+: 94.5 ± 3.7). Peripheral blood (PB) leukocytes were used as a control to Walk-Out lamina propria leukocytes (WO-LPL). If possible, PB and WO-LP leukocytes from the same donor were investigated. In some cases, due to logistic reasons, PB leukocytes from different, allogeneic donors were also tested. In contrast to WO-LPMO, peripheral blood monocytes (PBMO) do not express CD80 (Fig. 1B). Therefore, PBMO were activated with 1 µg/mL LPS for 8 h to induce CD80 expression before their introduction into the cultures to test RhuDex® (Fig. 1B, C). To exclude that T cells become activated by LPS, PB leukocytes were split into two fractions for differential treatment of T cells and monocytes before co-incubation. From fraction one, CD14+ monocytes were isolated and activated with LPS. Fraction two was placed in culture flasks for 8 h and subsequently the portion of PBL that had not adhered to plastic (non-adherent PBL, including T cells) was harvested. Cell composition and lack of strong T cell pre-activation in non-adherent PBL from allogeneic and autologous donors as well as in WO-LPL are reported in Fig. S1(A, B).


Immunomodulation of human intestinal T cells by the synthetic CD80 antagonist RhuDex®.

Heninger AK, Wentrup S, Al-Saeedi M, Schiessling S, Giese T, Wartha F, Meuer S, Schröder-Braunstein J - Immun Inflamm Dis (2014)

Expression of CD80 and CD86 on WO-LPL and PBMO. (A) Representative FACS plots of WO-LPL harvested after 36 h of organ culture and stained for surface expression of CD33 and CD14 (upper panel). Further, the surface expression of CD80 and CD86 of CD33+ WO-LPMO (lower panel) is shown. Numbers in each quadrant indicate %. (B) Peripheral blood monocytes (PBMO) were isolated from autologous PB using magnetic beads and activated with 1 µg/mL LPS for 8 h to induce CD80 expression. Representative FACS plots showing the purity of isolated CD14+CD33+ PBMO (upper panel) and their expression of CD80 in the absence or presence of LPS activation (lower panel). (C) CD80 (left panel) and CD86 (right panel) surface expression (%) of CD33+ WO-LPMO (7 tissue donors) and CD14+CD33+ PBMO (autologous: PB from 4 of the tissue donors; PB from 4 allogeneic donors).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257762&req=5

fig01: Expression of CD80 and CD86 on WO-LPL and PBMO. (A) Representative FACS plots of WO-LPL harvested after 36 h of organ culture and stained for surface expression of CD33 and CD14 (upper panel). Further, the surface expression of CD80 and CD86 of CD33+ WO-LPMO (lower panel) is shown. Numbers in each quadrant indicate %. (B) Peripheral blood monocytes (PBMO) were isolated from autologous PB using magnetic beads and activated with 1 µg/mL LPS for 8 h to induce CD80 expression. Representative FACS plots showing the purity of isolated CD14+CD33+ PBMO (upper panel) and their expression of CD80 in the absence or presence of LPS activation (lower panel). (C) CD80 (left panel) and CD86 (right panel) surface expression (%) of CD33+ WO-LPMO (7 tissue donors) and CD14+CD33+ PBMO (autologous: PB from 4 of the tissue donors; PB from 4 allogeneic donors).
Mentions: Because RhuDex® binds to CD80, we ensured the presence of CD80 on immunocompetent cells emigrating from our gut-culture model of general inflammation, following EDTA-mediated loss of the epithelial layer. As shown in Fig. 1(A, C) “Walk-Out” lamina propria myeloid cells (CD66b−CD33+ WO-LPMO) express high amounts of CD80 and CD86 (% CD80+: 91.3 ± 3.5; % CD86+: 94.5 ± 3.7). Peripheral blood (PB) leukocytes were used as a control to Walk-Out lamina propria leukocytes (WO-LPL). If possible, PB and WO-LP leukocytes from the same donor were investigated. In some cases, due to logistic reasons, PB leukocytes from different, allogeneic donors were also tested. In contrast to WO-LPMO, peripheral blood monocytes (PBMO) do not express CD80 (Fig. 1B). Therefore, PBMO were activated with 1 µg/mL LPS for 8 h to induce CD80 expression before their introduction into the cultures to test RhuDex® (Fig. 1B, C). To exclude that T cells become activated by LPS, PB leukocytes were split into two fractions for differential treatment of T cells and monocytes before co-incubation. From fraction one, CD14+ monocytes were isolated and activated with LPS. Fraction two was placed in culture flasks for 8 h and subsequently the portion of PBL that had not adhered to plastic (non-adherent PBL, including T cells) was harvested. Cell composition and lack of strong T cell pre-activation in non-adherent PBL from allogeneic and autologous donors as well as in WO-LPL are reported in Fig. S1(A, B).

Bottom Line: Results show that RhuDex® caused a profound reduction of LPL and PBL proliferation, while Abatacept (CTLA-4-Ig) inhibited LPL proliferation to a small degree, and had no effect on PBL proliferation.Furthermore, Abatacept significantly inhibited IL-2, TNF-α, and IFN-γ release from LPL, primarily produced by CD4(+) T cells, where IL-2 blockage was surprisingly strong, suggesting a down-regulating effect on regulatory T cells.Thus, RhuDex® efficiently inhibited lamina propria and peripheral blood T-cell activation in this pre-clinical study making it a promising drug candidate for the treatment of intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunology, University Hospital Heidelberg Im Neuenheimer Feld 305, 69120, Heidelberg, Germany.

ABSTRACT
Deregulated activation of mucosal lamina propria T cells plays a central role in the pathogenesis of intestinal inflammation. One of the means to attenuate T cell activation is by blocking the CD28/CD80 co-stimulatory pathway. Here we investigate RhuDex®, a small molecule that binds to human CD80, for its effects on the activation of lamina propria T cells employing a gut-culture model of inflammation. To this end, lamina propria leukocytes (LPL) and peripheral blood lymphocytes (PBL) were stimulated either through the CD3/T-cell-receptor complex or the CD2-receptor (CD2) employing agonistic monoclonal antibodies. Co-stimulatory signals were provided by CD80/CD86 present on lamina propria myeloid cells or LPS-activated peripheral blood monocytes. Results show that RhuDex® caused a profound reduction of LPL and PBL proliferation, while Abatacept (CTLA-4-Ig) inhibited LPL proliferation to a small degree, and had no effect on PBL proliferation. Furthermore, Abatacept significantly inhibited IL-2, TNF-α, and IFN-γ release from LPL, primarily produced by CD4(+) T cells, where IL-2 blockage was surprisingly strong, suggesting a down-regulating effect on regulatory T cells. In contrast, in the presence of RhuDex®, secretion of IL-17, again mostly by CD4(+) T cells, and IFN-γ was inhibited in LPL and PBL, yet IL-2 remained unaffected. Thus, RhuDex® efficiently inhibited lamina propria and peripheral blood T-cell activation in this pre-clinical study making it a promising drug candidate for the treatment of intestinal inflammation.

No MeSH data available.


Related in: MedlinePlus