The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the mai... more The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initi...
Murine CD4 T cells cultured under type 1 polarizing conditions selectively express significantly ... more Murine CD4 T cells cultured under type 1 polarizing conditions selectively express significantly higher levels of the very late antigen (VLA)-4 and VLA-6 integrins when compared with T cells cultured under type 2 or nonpolarizing (type 0) conditions. This difference appears due to the action of interleukin (IL)-4, as loss of VLA-4/-6 expression on Th cells was prevented by inclusion of neutralizing anti-IL-4 mAb during the initial culture period. We also observed that CD4 T cells deficient in Stat6, a critical component of the IL-4R signaling cascade, retained high levels of VLA-4 and VLA-6 expression, regardless of IL-4 status in the culture conditions. When applied to committed Th1 cells, rIL-4 readily inhibited VLA-4 and VLA-6 expression to levels observed for Th2 cells, without altering the type 1 functional status of these cells. Conversely, low levels of VLA-4/VLA-6 expressed by committed Th2 cells could not be resurrected by culture in the presence of the Th1-kines IL-12p70 and interferon-gamma. Predictably, among the Th populations evaluated, Th1 cells alone adhered efficiently to, and were costimulated by, plate-bound VCAM-1 and laminin in a VLA-4-dependent or VLA-6-dependent manner, respectively. Finally, adoptive-transferred Th1 (but not Th2) cells developed from OT-II mice were uniquely competent to traffick into OVA M05 melanoma lesions in vivo, thereby enhancing the therapeutic benefits associated with cotransferred OVA-specific type 1 CD8 (OT-I) cells. These data suggest that treatment strategies capable of sustaining/enhancing VLA-4/VLA-6 expression on Th1 effector cells may yield improved clinical efficacy in the cancer setting.
Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune syste... more Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of NK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4+ and CD8+ T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.
The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the mai... more The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initi...
Murine CD4 T cells cultured under type 1 polarizing conditions selectively express significantly ... more Murine CD4 T cells cultured under type 1 polarizing conditions selectively express significantly higher levels of the very late antigen (VLA)-4 and VLA-6 integrins when compared with T cells cultured under type 2 or nonpolarizing (type 0) conditions. This difference appears due to the action of interleukin (IL)-4, as loss of VLA-4/-6 expression on Th cells was prevented by inclusion of neutralizing anti-IL-4 mAb during the initial culture period. We also observed that CD4 T cells deficient in Stat6, a critical component of the IL-4R signaling cascade, retained high levels of VLA-4 and VLA-6 expression, regardless of IL-4 status in the culture conditions. When applied to committed Th1 cells, rIL-4 readily inhibited VLA-4 and VLA-6 expression to levels observed for Th2 cells, without altering the type 1 functional status of these cells. Conversely, low levels of VLA-4/VLA-6 expressed by committed Th2 cells could not be resurrected by culture in the presence of the Th1-kines IL-12p70 and interferon-gamma. Predictably, among the Th populations evaluated, Th1 cells alone adhered efficiently to, and were costimulated by, plate-bound VCAM-1 and laminin in a VLA-4-dependent or VLA-6-dependent manner, respectively. Finally, adoptive-transferred Th1 (but not Th2) cells developed from OT-II mice were uniquely competent to traffick into OVA M05 melanoma lesions in vivo, thereby enhancing the therapeutic benefits associated with cotransferred OVA-specific type 1 CD8 (OT-I) cells. These data suggest that treatment strategies capable of sustaining/enhancing VLA-4/VLA-6 expression on Th1 effector cells may yield improved clinical efficacy in the cancer setting.
Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune syste... more Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of NK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4+ and CD8+ T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.
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