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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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Related in: MedlinePlus

CD40L-Tri upregulates expression of costimulatory molecules on normal B cells. CD19+ B cells isolated from normal volunteers (n = 3) were cultured with various formulations of CD40L, including CD40L expressed on transfected cells (irradiated NIH3T3/tCD40L), a recombinant homotrimer with a short linker (shrtCD40L, gray symbols), and the test recombinant protein with a long flexible linker (CD40L-Tri, black symbols). Cells were evaluated by flow cytometry for expression of CD80, CD83, and CD86 at the indicated times. The mean MFI and error bars (±SD = standard deviation) for each treatment group are shown at each time point
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Fig2: CD40L-Tri upregulates expression of costimulatory molecules on normal B cells. CD19+ B cells isolated from normal volunteers (n = 3) were cultured with various formulations of CD40L, including CD40L expressed on transfected cells (irradiated NIH3T3/tCD40L), a recombinant homotrimer with a short linker (shrtCD40L, gray symbols), and the test recombinant protein with a long flexible linker (CD40L-Tri, black symbols). Cells were evaluated by flow cytometry for expression of CD80, CD83, and CD86 at the indicated times. The mean MFI and error bars (±SD = standard deviation) for each treatment group are shown at each time point

Mentions: Many of the immunostimulatory consequences of CD40L activation of B cells are mediated through its ability to upregulate expression of costimulatory molecules such as CD80, CD83, and CD86. To examine the potency of CD40L-Tri, we measured its ability to enhance expression of these molecules compared with known formulations of CD40L, namely the transfected murine fibroblast line (NIH3T3/tCD40L) and commercially available epitope-tagged homotrimeric forms that are formulated with a short linker (designated ‘shrtCD40L’) and that are inactive unless cross-linked by tag-specific antibodies. Culture of normal CD19+ B cells with CD40L-Tri (at 2 μg/ml; n = 3) consistently resulted in expression of CD80, CD83, and CD86 at 48 h with mean increases of 29-, 24-, and 165-fold, respectively, (p < 0.05) which was comparable to NIH3T3/tCD40L (25-, 22-, and 146-fold, respectively; p < 0.05) (Fig. 2). Seventy-two hours following exposure to CD40L-Tri (2 μg/ml), the expression of CD80 and CD86 persisted (mean of 34- and 180-fold) while expression of CD83 decreased to 13-fold increase from baseline. No increase in expression of costimulatory molecules was observed following exposure to shrtCD40L, as expected without cross-linking.Fig. 2


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 upregulates expression of costimulatory molecules on normal B cells. CD19+ B cells isolated from normal volunteers (n = 3) were cultured with various formulations of CD40L, including CD40L expressed on transfected cells (irradiated NIH3T3/tCD40L), a recombinant homotrimer with a short linker (shrtCD40L, gray symbols), and the test recombinant protein with a long flexible linker (CD40L-Tri, black symbols). Cells were evaluated by flow cytometry for expression of CD80, CD83, and CD86 at the indicated times. The mean MFI and error bars (±SD = standard deviation) for each treatment group are shown at each time point
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569584&req=5

Fig2: CD40L-Tri upregulates expression of costimulatory molecules on normal B cells. CD19+ B cells isolated from normal volunteers (n = 3) were cultured with various formulations of CD40L, including CD40L expressed on transfected cells (irradiated NIH3T3/tCD40L), a recombinant homotrimer with a short linker (shrtCD40L, gray symbols), and the test recombinant protein with a long flexible linker (CD40L-Tri, black symbols). Cells were evaluated by flow cytometry for expression of CD80, CD83, and CD86 at the indicated times. The mean MFI and error bars (±SD = standard deviation) for each treatment group are shown at each time point
Mentions: Many of the immunostimulatory consequences of CD40L activation of B cells are mediated through its ability to upregulate expression of costimulatory molecules such as CD80, CD83, and CD86. To examine the potency of CD40L-Tri, we measured its ability to enhance expression of these molecules compared with known formulations of CD40L, namely the transfected murine fibroblast line (NIH3T3/tCD40L) and commercially available epitope-tagged homotrimeric forms that are formulated with a short linker (designated ‘shrtCD40L’) and that are inactive unless cross-linked by tag-specific antibodies. Culture of normal CD19+ B cells with CD40L-Tri (at 2 μg/ml; n = 3) consistently resulted in expression of CD80, CD83, and CD86 at 48 h with mean increases of 29-, 24-, and 165-fold, respectively, (p < 0.05) which was comparable to NIH3T3/tCD40L (25-, 22-, and 146-fold, respectively; p < 0.05) (Fig. 2). Seventy-two hours following exposure to CD40L-Tri (2 μg/ml), the expression of CD80 and CD86 persisted (mean of 34- and 180-fold) while expression of CD83 decreased to 13-fold increase from baseline. No increase in expression of costimulatory molecules was observed following exposure to shrtCD40L, as expected without cross-linking.Fig. 2

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