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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 can effectively expand normal B cells. Average fold increase in B cell number, when CD19+ B cells were seeded at a high density (2 × 106 CD19+ B cells/2 ml) and b low density (0.3 × 106 CD19+/2 ml). B cells were incubated with the indicated CD40L formulation as described in ‘Materials and methods’, counted on the indicated days and the ratio of cell number to baseline was calculated, with each line representing the mean ± SD over time, obtained from testing of cells from 3 adult volunteers. In (a), the significance is only shown for day 28 based on modeling for each group versus IL-4 alone, and the trend over all time points was significantly different for each group versus IL-4, p < 0.001. **p < 0.001 for each CD40L-Tri group compared with IL-4 alone. c CFSE-labeled normal B cells were stimulated with CD40L-Tri and cell division measured by CFSE dilution (shaded) by flow cytometry, compared with non-activated cells (‘IL4 alone’, non-shaded)
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Fig5: CD40L-Tri can effectively expand normal B cells. Average fold increase in B cell number, when CD19+ B cells were seeded at a high density (2 × 106 CD19+ B cells/2 ml) and b low density (0.3 × 106 CD19+/2 ml). B cells were incubated with the indicated CD40L formulation as described in ‘Materials and methods’, counted on the indicated days and the ratio of cell number to baseline was calculated, with each line representing the mean ± SD over time, obtained from testing of cells from 3 adult volunteers. In (a), the significance is only shown for day 28 based on modeling for each group versus IL-4 alone, and the trend over all time points was significantly different for each group versus IL-4, p < 0.001. **p < 0.001 for each CD40L-Tri group compared with IL-4 alone. c CFSE-labeled normal B cells were stimulated with CD40L-Tri and cell division measured by CFSE dilution (shaded) by flow cytometry, compared with non-activated cells (‘IL4 alone’, non-shaded)

Mentions: The ability of human CD40L to persistently expand normal B cells has been described [7, 11] and establishes the potential of using this molecule to generate a renewable source of antigen-presenting cells from primary human cells. We initially compared the ability of CD40L-Tri and NIH3T3/tCD40L cells to induce and sustain proliferation of B cells in high-density cultures (2 million cells/2 ml initial seeding density). As expected, we observed a lack of expansion of CD19+ B cells in the presence of IL-4-containing media without CD40L, and a ninefold mean expansion with NIH3T3/tCD40L cells at day 28 (Fig. 5a). CD40L-Tri (1, 2, and 10 μg/ml, replenished every 3–4 days) stimulated B cells expanded by 4–5-fold at day 28 compared with IL-4 (0.10-fold) (p < 0.05); however, this was less than the expansion with NIH3T3/tCD40L cells. These results were not altered by addition of conditioned media from NIH3T3 cells together with CD40L-Tri into the culture media, excluding the idea that the NIH3T3 cells provided soluble factors that contributed to B cell proliferation (data not shown).Fig. 5


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 can effectively expand normal B cells. Average fold increase in B cell number, when CD19+ B cells were seeded at a high density (2 × 106 CD19+ B cells/2 ml) and b low density (0.3 × 106 CD19+/2 ml). B cells were incubated with the indicated CD40L formulation as described in ‘Materials and methods’, counted on the indicated days and the ratio of cell number to baseline was calculated, with each line representing the mean ± SD over time, obtained from testing of cells from 3 adult volunteers. In (a), the significance is only shown for day 28 based on modeling for each group versus IL-4 alone, and the trend over all time points was significantly different for each group versus IL-4, p < 0.001. **p < 0.001 for each CD40L-Tri group compared with IL-4 alone. c CFSE-labeled normal B cells were stimulated with CD40L-Tri and cell division measured by CFSE dilution (shaded) by flow cytometry, compared with non-activated cells (‘IL4 alone’, non-shaded)
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Related In: Results  -  Collection

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Fig5: CD40L-Tri can effectively expand normal B cells. Average fold increase in B cell number, when CD19+ B cells were seeded at a high density (2 × 106 CD19+ B cells/2 ml) and b low density (0.3 × 106 CD19+/2 ml). B cells were incubated with the indicated CD40L formulation as described in ‘Materials and methods’, counted on the indicated days and the ratio of cell number to baseline was calculated, with each line representing the mean ± SD over time, obtained from testing of cells from 3 adult volunteers. In (a), the significance is only shown for day 28 based on modeling for each group versus IL-4 alone, and the trend over all time points was significantly different for each group versus IL-4, p < 0.001. **p < 0.001 for each CD40L-Tri group compared with IL-4 alone. c CFSE-labeled normal B cells were stimulated with CD40L-Tri and cell division measured by CFSE dilution (shaded) by flow cytometry, compared with non-activated cells (‘IL4 alone’, non-shaded)
Mentions: The ability of human CD40L to persistently expand normal B cells has been described [7, 11] and establishes the potential of using this molecule to generate a renewable source of antigen-presenting cells from primary human cells. We initially compared the ability of CD40L-Tri and NIH3T3/tCD40L cells to induce and sustain proliferation of B cells in high-density cultures (2 million cells/2 ml initial seeding density). As expected, we observed a lack of expansion of CD19+ B cells in the presence of IL-4-containing media without CD40L, and a ninefold mean expansion with NIH3T3/tCD40L cells at day 28 (Fig. 5a). CD40L-Tri (1, 2, and 10 μg/ml, replenished every 3–4 days) stimulated B cells expanded by 4–5-fold at day 28 compared with IL-4 (0.10-fold) (p < 0.05); however, this was less than the expansion with NIH3T3/tCD40L cells. These results were not altered by addition of conditioned media from NIH3T3 cells together with CD40L-Tri into the culture media, excluding the idea that the NIH3T3 cells provided soluble factors that contributed to B cell proliferation (data not shown).Fig. 5

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