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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-activated CLL cells effectively elicit patient CD8+ T cell responses. a Tumor cells freshly isolated from CLL patients (n = 4) were directly cultured with CD40L-Tri, irradiated CD40L-expressing NIH3T3 cells (NIH3T3/tCD40L), or recombinant CD40L (shrtCD40L) (open bars) or initially cryopreserved, thawed, and then incubated with the various CD40L formulations (black bars). Forty-eight hours later, the expression of CD80, CD83, and CD86 was evaluated by flow cytometry. The mean MFI and error bars (±SD) for each treatment group are depicted. b PBMCs from a patient with chronic lymphocytic leukemia (CLL) before hematopoietic allogeneic stem cell transplantation (‘pre-HSCT’) (top) and following transplant (‘post-HSCT’) (bottom) were stimulated with CD40L-Tri-activated autologous CLL cells, and immunomagnetically selected CD8+ T cells were restimulated on ELISPOT assay with autologous CLL cells activated by the indicated CD40L formulation. The mean number of IFNγ-specific spots is displayed for each group
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Fig4: CD40L-Tri-activated CLL cells effectively elicit patient CD8+ T cell responses. a Tumor cells freshly isolated from CLL patients (n = 4) were directly cultured with CD40L-Tri, irradiated CD40L-expressing NIH3T3 cells (NIH3T3/tCD40L), or recombinant CD40L (shrtCD40L) (open bars) or initially cryopreserved, thawed, and then incubated with the various CD40L formulations (black bars). Forty-eight hours later, the expression of CD80, CD83, and CD86 was evaluated by flow cytometry. The mean MFI and error bars (±SD) for each treatment group are depicted. b PBMCs from a patient with chronic lymphocytic leukemia (CLL) before hematopoietic allogeneic stem cell transplantation (‘pre-HSCT’) (top) and following transplant (‘post-HSCT’) (bottom) were stimulated with CD40L-Tri-activated autologous CLL cells, and immunomagnetically selected CD8+ T cells were restimulated on ELISPOT assay with autologous CLL cells activated by the indicated CD40L formulation. The mean number of IFNγ-specific spots is displayed for each group

Mentions: We further tested the capacity of CD40L-Tri to increase the immunostimulatory activity of malignant B cells. Chronic lymphocytic leukemia (CLL), a disease of clonal CD19+ CD5+ CD23+ B cells, is sensitive to immune modulation. The development of methods to stimulate and monitor immunity against these leukemia cells, however, has been impaired by the poor antigen-presenting ability of CLL cells due to their low expression of costimulatory molecules. These defects can be reversed through stimulation with CD40L, for example, through use of the widely used NIH3T3/tCD40L system [23]. We observed that like normal CD19+ B cells, CLL cells (either fresh or cryopreserved) gain enhanced expression of costimulatory molecules following CD40L-Tri exposure (Fig. 4a, Supplemental Figure 2). We therefore tested a series of PBMC samples collected from CLL patients before and after allogeneic hematopoietic stem cell transplantation (HSCT) for the development of anti-CLL immunity after HSCT. These patients demonstrated molecular remission following HSCT. As shown in the example in Fig. 4b, patient PBMC that were collected 6 months following HSCT were co-cultured with CD40L-Tri-activated autologous CLL cells, and then purified CD8 T cells were restimulated in an IFNγ-ELISPOT assay with autologous CLL cells activated by CD40L-Tri, NIH3T3/tCD40L, or shrtCD40L, or by PHA. CD40L activation of CLL cells by the CD40L-transfected cell line or by CD40L-Tri elicited equivalently high reactivity, while the shrtCD40L-activated tumor targets elicited detectable but weaker reactivity. Patient PBMC collected before HSCT had minimal reactivity.Fig. 4


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-activated CLL cells effectively elicit patient CD8+ T cell responses. a Tumor cells freshly isolated from CLL patients (n = 4) were directly cultured with CD40L-Tri, irradiated CD40L-expressing NIH3T3 cells (NIH3T3/tCD40L), or recombinant CD40L (shrtCD40L) (open bars) or initially cryopreserved, thawed, and then incubated with the various CD40L formulations (black bars). Forty-eight hours later, the expression of CD80, CD83, and CD86 was evaluated by flow cytometry. The mean MFI and error bars (±SD) for each treatment group are depicted. b PBMCs from a patient with chronic lymphocytic leukemia (CLL) before hematopoietic allogeneic stem cell transplantation (‘pre-HSCT’) (top) and following transplant (‘post-HSCT’) (bottom) were stimulated with CD40L-Tri-activated autologous CLL cells, and immunomagnetically selected CD8+ T cells were restimulated on ELISPOT assay with autologous CLL cells activated by the indicated CD40L formulation. The mean number of IFNγ-specific spots is displayed for each group
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Related In: Results  -  Collection

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Fig4: CD40L-Tri-activated CLL cells effectively elicit patient CD8+ T cell responses. a Tumor cells freshly isolated from CLL patients (n = 4) were directly cultured with CD40L-Tri, irradiated CD40L-expressing NIH3T3 cells (NIH3T3/tCD40L), or recombinant CD40L (shrtCD40L) (open bars) or initially cryopreserved, thawed, and then incubated with the various CD40L formulations (black bars). Forty-eight hours later, the expression of CD80, CD83, and CD86 was evaluated by flow cytometry. The mean MFI and error bars (±SD) for each treatment group are depicted. b PBMCs from a patient with chronic lymphocytic leukemia (CLL) before hematopoietic allogeneic stem cell transplantation (‘pre-HSCT’) (top) and following transplant (‘post-HSCT’) (bottom) were stimulated with CD40L-Tri-activated autologous CLL cells, and immunomagnetically selected CD8+ T cells were restimulated on ELISPOT assay with autologous CLL cells activated by the indicated CD40L formulation. The mean number of IFNγ-specific spots is displayed for each group
Mentions: We further tested the capacity of CD40L-Tri to increase the immunostimulatory activity of malignant B cells. Chronic lymphocytic leukemia (CLL), a disease of clonal CD19+ CD5+ CD23+ B cells, is sensitive to immune modulation. The development of methods to stimulate and monitor immunity against these leukemia cells, however, has been impaired by the poor antigen-presenting ability of CLL cells due to their low expression of costimulatory molecules. These defects can be reversed through stimulation with CD40L, for example, through use of the widely used NIH3T3/tCD40L system [23]. We observed that like normal CD19+ B cells, CLL cells (either fresh or cryopreserved) gain enhanced expression of costimulatory molecules following CD40L-Tri exposure (Fig. 4a, Supplemental Figure 2). We therefore tested a series of PBMC samples collected from CLL patients before and after allogeneic hematopoietic stem cell transplantation (HSCT) for the development of anti-CLL immunity after HSCT. These patients demonstrated molecular remission following HSCT. As shown in the example in Fig. 4b, patient PBMC that were collected 6 months following HSCT were co-cultured with CD40L-Tri-activated autologous CLL cells, and then purified CD8 T cells were restimulated in an IFNγ-ELISPOT assay with autologous CLL cells activated by CD40L-Tri, NIH3T3/tCD40L, or shrtCD40L, or by PHA. CD40L activation of CLL cells by the CD40L-transfected cell line or by CD40L-Tri elicited equivalently high reactivity, while the shrtCD40L-activated tumor targets elicited detectable but weaker reactivity. Patient PBMC collected before HSCT had minimal reactivity.Fig. 4

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