- Email: [email protected]
Creation of novel costimulatory ligands through directed molecular evolution
568
News & Comment
TRENDS in Immunology Vol.23 No.12 December 2002
Journal Club
Heat-shock protein receptor debate hots up Heat-shock proteins (HSPs) have come a long way from their original description as proteins induced by heat shock involved in the puffing of chromosomes in Drosophila. They are now viewed as key players in the immune response as integrators of the innate and acquired immune responses [1]. Although initial attention on the role of HSPs in the immune response focused on their non-specific ‘adjuvant’ properties, more recent studies have highlighted their role as ‘antigen-carriers’ in the cross-priming of MHC class I molecules. This is thought to be a consequence of their normal cellular function as molecular chaperones, and is particularly relevant to the integration of innate and acquired immunity because antigen capture is central to the induction of the acquired immune response [1]. Much current work has therefore focused on the identification of the cellular receptors for HSPs and in particular those receptors that were present on antigen-presenting cells (APCs) of the immune system. Although initial work implicated the macrophage scavenger receptor as a receptor for HSPs, the identification of CD91 as the receptor for the multifunctional HSP, grp96, has led to its proposal as the primary HSP receptor involved in the phenomenon of cross-priming. However, the cellular
distribution of CD91 and, in particular, equivocal data of its expression on the most professional of APCs, dendritic cells (DCs), has led to much debate on the nature of the ‘true’ HSP receptor. Becker et al. [2] now enter the debate with the identification of that well known APC surface molecule CD40 as the receptor for human HSP70–peptide complexes and in particular for the capture of chaperoned antigenic peptides for cross-priming of MHC class I molecules. The authors demonstrate that CD40 binds the N-terminal ATPase domain of HSP70 at a site that overlaps the binding site for the co-chaperone hip that has previously been shown to stabilize peptide binding to HSP70. These studies are, however, in direct contrast to earlier studies identifying CD40 as the cellular receptor for the bacterial analogue of HSP70 DnaK but not human HSP70, which also implicated CD14 as the receptor for HSP70. The resolution of this discrepancy appears to be in the domain structure of the HSP70 molecule. The present studies show that the binding of the ATPase domain by CD40 appears to be restricted to the ADP-bound state, which means that CD40 only recognizes HSP70 complexed to antigenic peptide [2]. By contrast, the previous studies were done in the absence of nucleotide or
peptide binding and indeed the binding of CD40 by DnaK was localized to the C-terminal domain of the molecule. Most intriguingly, the C-terminal domain of both human and bacterial HSP70 has been implicated in innate immunity with the induction of chemokine release by cells of the innate immune system. It is, therefore, probable that HSPs and HSP–peptide complexes bind distinct receptors with distinct functional consequences reflecting the role of HSPs in both innate and acquired immune responses. This proposal is consistent with the recent demonstration of HSP binding to the Toll-like receptors (TLRs), TLR2 and TLR4, which reinforces the suggestion that pathogen-specific information might be carried not just by HSP-chaperoned antigens but by the HSPs themselves [1]. 1 Colaco, C. (1998) Towards a unified theory of immunity: dendritic cells, stress proteins and antigen capture. Cell Mol. Biol. 44, 883–890 2 Becker, T. et al. (2002) CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes. J. Cell Biol. 158, 1277–1285
Camilo Colaco [email protected]
Creation of novel costimulatory ligands through directed molecular evolution In physiologically relevant situations, activation of naïve T cells is thought to require two signals, an antigen-specific signal mediated through the T-cell receptor (signal 1) and an antigen-independent costimulatory signal mediated through activating receptors also expressed on T cells (signal 2). The best studied activating receptor, CD28, is able to costimulate T cells through engagement of either the CD80 (B7-1) or CD86 (B7-2) costimulatory ligand, both of which are expressed on professional antigen-presenting cells (APCs). Although able to augment T-cell activation, CD80 and CD86 can also paradoxically deliver http://immunology.trends.com
negative regulatory signals to the same T cells through engagement of the inhibitory receptor, CTLA-4 (CD152), which is upregulated on activated T cells. This highly regulated costimulatory mechanism is undoubtedly of biological value, however, the ability of the CD80 and CD86 to simultaneously promote and suppress the immune response presents particular difficulties in their use for the development of immune-based therapies. In an innovative approach to circumvent this problem, Lazetic et al. used directed molecular evolution to create chimeric molecules that selectively interact with
either only CD28 or only CTLA-4 [1]. Specifically, the authors isolated several mammalian cDNA homologs encoding CD80 (these related genes had 57–99% sequence identity) and used DNA shuffling to create a library of chimeric genes. By using a flow cytometric based screen and selecting those chimeras with either enhanced binding to CD28 or CTLA-4, and repeating this process of DNA shuffling and screening, the authors were able to isolate two novel chimeras with exclusive binding to either CD28 or CTLA-4. The authors called these chimeras CD28 binding protein (CD28BP) and CTLA-4 binding protein (CTLA-4 BP), respectively.
1471-4906/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
Importantly, the binding properties of these novel proteins were unique and not shared by any of the parental genes. As a final step, the authors assessed the functional ability of these chimeric proteins. When human T cells were stimulated in the presence of transfected cells expressing either of these chimeric proteins, CD28BP selectively augmented T-cell proliferation and interferon-γ (IFN-γ) production, however, CTLA-4BP suppressed T-cell proliferation and enhanced interleukin-10 (IL-10) production. These properties are consistent with CD28BP and CTLA-4BP selectively acting as ligands for CD28 and CTLA-4, respectively. The chimeric molecules created by Lazetic et al. offer a number of potential advantages over currently used approaches to augment or suppress T-cell-mediated immune responses using
TRENDS in Immunology Vol.23 No.12 December 2002
the CD28–CTLA-4 pathway. For example, several groups have sought to augment T-cell-mediated responses by providing exogenously expressed CD80 or ‘These properties are consistent with CD28BP and CTLA-4BP selectively acting as ligands for CD28 and CTLA-4, respectively.’ CD86 to APCs. Although there is clear evidence for the efficacy of this approach, this also results in the unintended consequence of augmenting signalling through the inhibitory CTLA-4 receptor. CD28BP could have an obvious advantage in this situation. It will also be of interest to evaluate the ability of CTLA-4BP to suppress T-cell-mediated immune responses. In contrast to the use of soluble antibodies (and other reagents), CTLA-4BP could be directly expressed on cells
569
coexpressing specific antigens, thereby offering a preferential means for selectively downregulating the immune response against a defined set of antigens. Although the results reported by Lazetic et al. provide the framework for evaluating these intriguing possibilities, perhaps of equal excitement is the potential to use this approach for optimizing the properties of a growing number of newly identified costimulatory molecules. 1 Lazetic, S. et al. (2002) Chimeric costimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells. J. Biol. Chem. 41, 38660–38668
Jan Woraratanadharm [email protected] Mark Rubinstein [email protected]
Vaccination against Alzheimer’s disease: hope renewed Alzheimer’s disease (AD) is characterized by progressive dementia, probably due to the accumulation of amyloid plaques in the brain. These plaques essentially consist of peptides [Aβ1–40 and Aβ1–42 (Aβ1–40/42)] derived from the amyloid precursor protein (APP). There is currently little symptomatic treatment and no curative therapy available, which makes AD one of the most distressing illnesses for both the patients and their families. A ray of hope for a possible therapy was provided recently by vaccination experiments in transgenic mice. Specifically, mice expressing mutant forms of human APP efficiently cleaved into Aβ1–40/42 peptides developed AD-like pathology. Surprisingly, both plaque burden and dementia could be reduced in these mice by active immunization for antibody induction using Aβ1–42 formulated in adjuvants as a vaccine (AN1792). Although the mechanism of plaque removal remains unclear, vaccination has proved to be efficient in a variety of APP-transgenic mouse models. Progress towards the clinic was rapid and ELAN pharmaceuticals, the front runner in the field, initiated a Phase II trial designed to demonstrate the efficacy of the therapy in humans. However, it was this particular study that brought hopes and progress to an abrupt http://immunology.trends.com
halt because a few treated patients developed signs of aseptic encephalitis or meningitis following the second administration of the Aβ1–42 vaccine. Although little information has been released publicly, the symptoms were consistent with the induction of autoimmunity, an inherent risk and concern when vaccinating against self-antigens, such as Aβ1–42. Therefore this trial has been stopped. A detailed understanding of the immune responses induced in the vaccinated individuals is now crucial for further development of vaccination strategies that avoid these severe (albeit treatable) side-effects. It is of particular importance to understand the origin of the vaccine-induced disease, namely was it non-specifically caused by the adjuvants used, were the antibodies precipitating inflammation or were Aβ1-42-specific T cells responsible for the side-effects. Thankfully, the study by Hock et al. [1] now offers the first insights into these questions. Of the 24 patients vaccinated in the study center in Zurich, almost all raised a significant antibody response. In addition, the antibodies recognized plaques of transgenic mice and patients, a requirement for plaque-removal in the mouse model. However, in one individual, aseptic encephalitis
(meningitis) developed. Intriguingly, this particular patient had only raised a moderate antibody response. This observation indicates that specific antibodies alone might not be sufficient to cause disease in AD patients. Furthermore, the results are compatible with the view that T cells, rather than antibodies, are the major source of inflammation. This hypothesis is consistent with the fact that most, if not all, known inflammatory responses in the brain are caused by T cells rather than antibodies. Although it is obviously impossible to draw firm conclusions from a single patient, the data nevertheless suggest that vaccines against AD should aim at circumventing the induction of inflammatory T cell responses. Indeed, avoiding the use of adjuvants and restricting the size of the epitopes used for vaccination might offer valuable solutions for the generation of a viable vaccine against one of the most devastating diseases afflicting mankind. 1 Hock, C. et al. (2002) Generation of antibodies specific for β-amyloid by vaccination of patients with Alzheimer disease. Nat. Med. 10.1038/nm783 (http://www.nature.com/nm/)
Martin F. Bachmann [email protected]
1471-4906/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Immunology Vol.23 No.12 December 2002
Journal Club
Heat-shock protein receptor debate hots up Heat-shock proteins (HSPs) have come a long way from their original description as proteins induced by heat shock involved in the puffing of chromosomes in Drosophila. They are now viewed as key players in the immune response as integrators of the innate and acquired immune responses [1]. Although initial attention on the role of HSPs in the immune response focused on their non-specific ‘adjuvant’ properties, more recent studies have highlighted their role as ‘antigen-carriers’ in the cross-priming of MHC class I molecules. This is thought to be a consequence of their normal cellular function as molecular chaperones, and is particularly relevant to the integration of innate and acquired immunity because antigen capture is central to the induction of the acquired immune response [1]. Much current work has therefore focused on the identification of the cellular receptors for HSPs and in particular those receptors that were present on antigen-presenting cells (APCs) of the immune system. Although initial work implicated the macrophage scavenger receptor as a receptor for HSPs, the identification of CD91 as the receptor for the multifunctional HSP, grp96, has led to its proposal as the primary HSP receptor involved in the phenomenon of cross-priming. However, the cellular
distribution of CD91 and, in particular, equivocal data of its expression on the most professional of APCs, dendritic cells (DCs), has led to much debate on the nature of the ‘true’ HSP receptor. Becker et al. [2] now enter the debate with the identification of that well known APC surface molecule CD40 as the receptor for human HSP70–peptide complexes and in particular for the capture of chaperoned antigenic peptides for cross-priming of MHC class I molecules. The authors demonstrate that CD40 binds the N-terminal ATPase domain of HSP70 at a site that overlaps the binding site for the co-chaperone hip that has previously been shown to stabilize peptide binding to HSP70. These studies are, however, in direct contrast to earlier studies identifying CD40 as the cellular receptor for the bacterial analogue of HSP70 DnaK but not human HSP70, which also implicated CD14 as the receptor for HSP70. The resolution of this discrepancy appears to be in the domain structure of the HSP70 molecule. The present studies show that the binding of the ATPase domain by CD40 appears to be restricted to the ADP-bound state, which means that CD40 only recognizes HSP70 complexed to antigenic peptide [2]. By contrast, the previous studies were done in the absence of nucleotide or
peptide binding and indeed the binding of CD40 by DnaK was localized to the C-terminal domain of the molecule. Most intriguingly, the C-terminal domain of both human and bacterial HSP70 has been implicated in innate immunity with the induction of chemokine release by cells of the innate immune system. It is, therefore, probable that HSPs and HSP–peptide complexes bind distinct receptors with distinct functional consequences reflecting the role of HSPs in both innate and acquired immune responses. This proposal is consistent with the recent demonstration of HSP binding to the Toll-like receptors (TLRs), TLR2 and TLR4, which reinforces the suggestion that pathogen-specific information might be carried not just by HSP-chaperoned antigens but by the HSPs themselves [1]. 1 Colaco, C. (1998) Towards a unified theory of immunity: dendritic cells, stress proteins and antigen capture. Cell Mol. Biol. 44, 883–890 2 Becker, T. et al. (2002) CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes. J. Cell Biol. 158, 1277–1285
Camilo Colaco [email protected]
Creation of novel costimulatory ligands through directed molecular evolution In physiologically relevant situations, activation of naïve T cells is thought to require two signals, an antigen-specific signal mediated through the T-cell receptor (signal 1) and an antigen-independent costimulatory signal mediated through activating receptors also expressed on T cells (signal 2). The best studied activating receptor, CD28, is able to costimulate T cells through engagement of either the CD80 (B7-1) or CD86 (B7-2) costimulatory ligand, both of which are expressed on professional antigen-presenting cells (APCs). Although able to augment T-cell activation, CD80 and CD86 can also paradoxically deliver http://immunology.trends.com
negative regulatory signals to the same T cells through engagement of the inhibitory receptor, CTLA-4 (CD152), which is upregulated on activated T cells. This highly regulated costimulatory mechanism is undoubtedly of biological value, however, the ability of the CD80 and CD86 to simultaneously promote and suppress the immune response presents particular difficulties in their use for the development of immune-based therapies. In an innovative approach to circumvent this problem, Lazetic et al. used directed molecular evolution to create chimeric molecules that selectively interact with
either only CD28 or only CTLA-4 [1]. Specifically, the authors isolated several mammalian cDNA homologs encoding CD80 (these related genes had 57–99% sequence identity) and used DNA shuffling to create a library of chimeric genes. By using a flow cytometric based screen and selecting those chimeras with either enhanced binding to CD28 or CTLA-4, and repeating this process of DNA shuffling and screening, the authors were able to isolate two novel chimeras with exclusive binding to either CD28 or CTLA-4. The authors called these chimeras CD28 binding protein (CD28BP) and CTLA-4 binding protein (CTLA-4 BP), respectively.
1471-4906/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
Importantly, the binding properties of these novel proteins were unique and not shared by any of the parental genes. As a final step, the authors assessed the functional ability of these chimeric proteins. When human T cells were stimulated in the presence of transfected cells expressing either of these chimeric proteins, CD28BP selectively augmented T-cell proliferation and interferon-γ (IFN-γ) production, however, CTLA-4BP suppressed T-cell proliferation and enhanced interleukin-10 (IL-10) production. These properties are consistent with CD28BP and CTLA-4BP selectively acting as ligands for CD28 and CTLA-4, respectively. The chimeric molecules created by Lazetic et al. offer a number of potential advantages over currently used approaches to augment or suppress T-cell-mediated immune responses using
TRENDS in Immunology Vol.23 No.12 December 2002
the CD28–CTLA-4 pathway. For example, several groups have sought to augment T-cell-mediated responses by providing exogenously expressed CD80 or ‘These properties are consistent with CD28BP and CTLA-4BP selectively acting as ligands for CD28 and CTLA-4, respectively.’ CD86 to APCs. Although there is clear evidence for the efficacy of this approach, this also results in the unintended consequence of augmenting signalling through the inhibitory CTLA-4 receptor. CD28BP could have an obvious advantage in this situation. It will also be of interest to evaluate the ability of CTLA-4BP to suppress T-cell-mediated immune responses. In contrast to the use of soluble antibodies (and other reagents), CTLA-4BP could be directly expressed on cells
569
coexpressing specific antigens, thereby offering a preferential means for selectively downregulating the immune response against a defined set of antigens. Although the results reported by Lazetic et al. provide the framework for evaluating these intriguing possibilities, perhaps of equal excitement is the potential to use this approach for optimizing the properties of a growing number of newly identified costimulatory molecules. 1 Lazetic, S. et al. (2002) Chimeric costimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells. J. Biol. Chem. 41, 38660–38668
Jan Woraratanadharm [email protected] Mark Rubinstein [email protected]
Vaccination against Alzheimer’s disease: hope renewed Alzheimer’s disease (AD) is characterized by progressive dementia, probably due to the accumulation of amyloid plaques in the brain. These plaques essentially consist of peptides [Aβ1–40 and Aβ1–42 (Aβ1–40/42)] derived from the amyloid precursor protein (APP). There is currently little symptomatic treatment and no curative therapy available, which makes AD one of the most distressing illnesses for both the patients and their families. A ray of hope for a possible therapy was provided recently by vaccination experiments in transgenic mice. Specifically, mice expressing mutant forms of human APP efficiently cleaved into Aβ1–40/42 peptides developed AD-like pathology. Surprisingly, both plaque burden and dementia could be reduced in these mice by active immunization for antibody induction using Aβ1–42 formulated in adjuvants as a vaccine (AN1792). Although the mechanism of plaque removal remains unclear, vaccination has proved to be efficient in a variety of APP-transgenic mouse models. Progress towards the clinic was rapid and ELAN pharmaceuticals, the front runner in the field, initiated a Phase II trial designed to demonstrate the efficacy of the therapy in humans. However, it was this particular study that brought hopes and progress to an abrupt http://immunology.trends.com
halt because a few treated patients developed signs of aseptic encephalitis or meningitis following the second administration of the Aβ1–42 vaccine. Although little information has been released publicly, the symptoms were consistent with the induction of autoimmunity, an inherent risk and concern when vaccinating against self-antigens, such as Aβ1–42. Therefore this trial has been stopped. A detailed understanding of the immune responses induced in the vaccinated individuals is now crucial for further development of vaccination strategies that avoid these severe (albeit treatable) side-effects. It is of particular importance to understand the origin of the vaccine-induced disease, namely was it non-specifically caused by the adjuvants used, were the antibodies precipitating inflammation or were Aβ1-42-specific T cells responsible for the side-effects. Thankfully, the study by Hock et al. [1] now offers the first insights into these questions. Of the 24 patients vaccinated in the study center in Zurich, almost all raised a significant antibody response. In addition, the antibodies recognized plaques of transgenic mice and patients, a requirement for plaque-removal in the mouse model. However, in one individual, aseptic encephalitis
(meningitis) developed. Intriguingly, this particular patient had only raised a moderate antibody response. This observation indicates that specific antibodies alone might not be sufficient to cause disease in AD patients. Furthermore, the results are compatible with the view that T cells, rather than antibodies, are the major source of inflammation. This hypothesis is consistent with the fact that most, if not all, known inflammatory responses in the brain are caused by T cells rather than antibodies. Although it is obviously impossible to draw firm conclusions from a single patient, the data nevertheless suggest that vaccines against AD should aim at circumventing the induction of inflammatory T cell responses. Indeed, avoiding the use of adjuvants and restricting the size of the epitopes used for vaccination might offer valuable solutions for the generation of a viable vaccine against one of the most devastating diseases afflicting mankind. 1 Hock, C. et al. (2002) Generation of antibodies specific for β-amyloid by vaccination of patients with Alzheimer disease. Nat. Med. 10.1038/nm783 (http://www.nature.com/nm/)
Martin F. Bachmann [email protected]
1471-4906/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.