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CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

Naito M, Hainz U, Burkhardt UE, Fu B, Ahove D, Stevenson KE, Rajasagi M, Zhu B, Alonso A, Witten E, Matsuoka K, Neuberg D, Duke-Cohan JS, Wu CJ, Freeman GJ - Cancer Immunol. Immunother. (2012)

Bottom Line: Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers.Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells.The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

View Article: PubMed Central - PubMed

Affiliation: Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

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CD40L-Tri generates activated B cells that strongly stimulate expansion of allogeneic CD8+ and CD4+ T cells. CD19+ B cells were cultured without CD40L (‘no CD40L’) or with various formulations of CD40L, including a recombinant homotrimer with a long flexible linker (‘CD40L-Tri’), a recombinant homotrimer with a short linker (‘shrtCD40L’), a recombinant 4-trimer form (‘Ultra CD40L’), or irradiated NIH3T3/tCD40L. Activated B cells were harvested after 2 days, irradiated, and co-cultured with allogeneic CD8+ T cells (a) or CD4+ T cells (b) at the indicated ratios. T cell proliferation was measured after 5 days. The figure displays the mean and error bars (+SD) for each treatment group from 3 adult volunteers assayed in triplicate. c Representative results (of experiments from 3 donors) of generation of M1 peptide-specific CD8+ T cells from a HLA-A2+ donor, by stimulation of T cells with M1 peptide (10 μg/ml) loaded on T2 cells, CD40L-Tri-activated B cells, normal B cells, or DCs. After 1 week, expanded CD8+ T cells (5,000) were co-cultured with the indicated target cells with or without M1 peptide for 24 h on IFNγ ELISPOT plates in triplicate
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Fig3: CD40L-Tri generates activated B cells that strongly stimulate expansion of allogeneic CD8+ and CD4+ T cells. CD19+ B cells were cultured without CD40L (‘no CD40L’) or with various formulations of CD40L, including a recombinant homotrimer with a long flexible linker (‘CD40L-Tri’), a recombinant homotrimer with a short linker (‘shrtCD40L’), a recombinant 4-trimer form (‘Ultra CD40L’), or irradiated NIH3T3/tCD40L. Activated B cells were harvested after 2 days, irradiated, and co-cultured with allogeneic CD8+ T cells (a) or CD4+ T cells (b) at the indicated ratios. T cell proliferation was measured after 5 days. The figure displays the mean and error bars (+SD) for each treatment group from 3 adult volunteers assayed in triplicate. c Representative results (of experiments from 3 donors) of generation of M1 peptide-specific CD8+ T cells from a HLA-A2+ donor, by stimulation of T cells with M1 peptide (10 μg/ml) loaded on T2 cells, CD40L-Tri-activated B cells, normal B cells, or DCs. After 1 week, expanded CD8+ T cells (5,000) were co-cultured with the indicated target cells with or without M1 peptide for 24 h on IFNγ ELISPOT plates in triplicate

Mentions: Since CD40L-Tri clearly induced expression of costimulatory molecules on B cells, we next investigated the capacity of CD40L-Tri-activated B cells to stimulate proliferation of allogeneic T cells. B cells were activated with CD40L-Tri or other CD40L formulations (shrt-CD40L, NIH3T3/tCD40L, Ultra-CD40L), irradiated, and co-cultured at a ratio of 2:1 or 10:1 with allogeneic CD4+ or CD8+ T cells. T cell proliferation was measured by [3H] thymidine incorporation at 5 days following the initiation of co-culture. CD40L-Tri-activated B cells (used at 1–2 μg/ml) consistently stimulated proliferation of CD8+ T cells from three normal volunteers (Fig. 3a, right panel). At a 2:1 ratio, CD8+ T cells, when exposed to allogeneic CD40L-Tri-activated B cells, proliferated 8–11-fold more than when exposed to non-activated B cells (p < 0.05), and 6–8-fold more than when exposed to B cells treated with shrt-CD40L (1 μg/ml) (p < 0.05), which is known to be non-bioactive in the absence of a cross-linking antibody. These results compared favorably to the extent of proliferation elicited by NIH3T3/tCD40L-activated B cells (22-fold more than non-activated B cells; p < 0.05). Similarly, as shown in Fig. 3b (right panel), we observed high proliferation of allogeneic CD4+ cells when co-cultured with irradiated CD40L-Tri-activated B cells at 2:1 ratio (12–13-fold, compared with either shrt-CD40L or non-activated B cells) that was comparable to stimulation by NIH3T3/tCD40L-activated B cells (14-fold). We also established that the stimulatory capacity of CD40L-Tri-activated B cells was as strong as B cells activated by a multimerized CD40L (Ultra-CD40L) [11] (Fig. 3a, b left panel). Together, these results demonstrate that CD40L-Tri has potent immunostimulatory activity.Fig. 3


CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

Naito M, Hainz U, Burkhardt UE, Fu B, Ahove D, Stevenson KE, Rajasagi M, Zhu B, Alonso A, Witten E, Matsuoka K, Neuberg D, Duke-Cohan JS, Wu CJ, Freeman GJ - Cancer Immunol. Immunother. (2012)

CD40L-Tri generates activated B cells that strongly stimulate expansion of allogeneic CD8+ and CD4+ T cells. CD19+ B cells were cultured without CD40L (‘no CD40L’) or with various formulations of CD40L, including a recombinant homotrimer with a long flexible linker (‘CD40L-Tri’), a recombinant homotrimer with a short linker (‘shrtCD40L’), a recombinant 4-trimer form (‘Ultra CD40L’), or irradiated NIH3T3/tCD40L. Activated B cells were harvested after 2 days, irradiated, and co-cultured with allogeneic CD8+ T cells (a) or CD4+ T cells (b) at the indicated ratios. T cell proliferation was measured after 5 days. The figure displays the mean and error bars (+SD) for each treatment group from 3 adult volunteers assayed in triplicate. c Representative results (of experiments from 3 donors) of generation of M1 peptide-specific CD8+ T cells from a HLA-A2+ donor, by stimulation of T cells with M1 peptide (10 μg/ml) loaded on T2 cells, CD40L-Tri-activated B cells, normal B cells, or DCs. After 1 week, expanded CD8+ T cells (5,000) were co-cultured with the indicated target cells with or without M1 peptide for 24 h on IFNγ ELISPOT plates in triplicate
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Related In: Results  -  Collection

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Fig3: CD40L-Tri generates activated B cells that strongly stimulate expansion of allogeneic CD8+ and CD4+ T cells. CD19+ B cells were cultured without CD40L (‘no CD40L’) or with various formulations of CD40L, including a recombinant homotrimer with a long flexible linker (‘CD40L-Tri’), a recombinant homotrimer with a short linker (‘shrtCD40L’), a recombinant 4-trimer form (‘Ultra CD40L’), or irradiated NIH3T3/tCD40L. Activated B cells were harvested after 2 days, irradiated, and co-cultured with allogeneic CD8+ T cells (a) or CD4+ T cells (b) at the indicated ratios. T cell proliferation was measured after 5 days. The figure displays the mean and error bars (+SD) for each treatment group from 3 adult volunteers assayed in triplicate. c Representative results (of experiments from 3 donors) of generation of M1 peptide-specific CD8+ T cells from a HLA-A2+ donor, by stimulation of T cells with M1 peptide (10 μg/ml) loaded on T2 cells, CD40L-Tri-activated B cells, normal B cells, or DCs. After 1 week, expanded CD8+ T cells (5,000) were co-cultured with the indicated target cells with or without M1 peptide for 24 h on IFNγ ELISPOT plates in triplicate
Mentions: Since CD40L-Tri clearly induced expression of costimulatory molecules on B cells, we next investigated the capacity of CD40L-Tri-activated B cells to stimulate proliferation of allogeneic T cells. B cells were activated with CD40L-Tri or other CD40L formulations (shrt-CD40L, NIH3T3/tCD40L, Ultra-CD40L), irradiated, and co-cultured at a ratio of 2:1 or 10:1 with allogeneic CD4+ or CD8+ T cells. T cell proliferation was measured by [3H] thymidine incorporation at 5 days following the initiation of co-culture. CD40L-Tri-activated B cells (used at 1–2 μg/ml) consistently stimulated proliferation of CD8+ T cells from three normal volunteers (Fig. 3a, right panel). At a 2:1 ratio, CD8+ T cells, when exposed to allogeneic CD40L-Tri-activated B cells, proliferated 8–11-fold more than when exposed to non-activated B cells (p < 0.05), and 6–8-fold more than when exposed to B cells treated with shrt-CD40L (1 μg/ml) (p < 0.05), which is known to be non-bioactive in the absence of a cross-linking antibody. These results compared favorably to the extent of proliferation elicited by NIH3T3/tCD40L-activated B cells (22-fold more than non-activated B cells; p < 0.05). Similarly, as shown in Fig. 3b (right panel), we observed high proliferation of allogeneic CD4+ cells when co-cultured with irradiated CD40L-Tri-activated B cells at 2:1 ratio (12–13-fold, compared with either shrt-CD40L or non-activated B cells) that was comparable to stimulation by NIH3T3/tCD40L-activated B cells (14-fold). We also established that the stimulatory capacity of CD40L-Tri-activated B cells was as strong as B cells activated by a multimerized CD40L (Ultra-CD40L) [11] (Fig. 3a, b left panel). Together, these results demonstrate that CD40L-Tri has potent immunostimulatory activity.Fig. 3

Bottom Line: Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers.Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells.The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

View Article: PubMed Central - PubMed

Affiliation: Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

Show MeSH
Related in: MedlinePlus