This article will describe coordinated analyses of how amino acid substitutions in the HLA class ... more This article will describe coordinated analyses of how amino acid substitutions in the HLA class I antigen binding groove modify chaperone interaction and peptide ligand presentation. By parallel testing of ligand presentation and chaperone interaction with a series of natural HLA-B subtypes, this study has discovered that position 116 of the HLA-B15 class I heavy chain is pivotal in both peptide selection and control of interaction between the assembly complex and the class I heavy chain. Correlated with these qualitative differences in peptide selection and chaperone association are quantitative differences in the expression levels of the HLA molecules at the cell surface. These parallel studies, therefore, demonstrate that particular HLA class I polymorphisms can simultaneously influence ligand presentation and interaction with intracellular chaperones.
Dendritic cells are well known for their potent ability, when fully differentiated or &am... more Dendritic cells are well known for their potent ability, when fully differentiated or 'mature', to stimulate immune responses to antigens they present efficiently to T cells. Mature DC have been used as experimental cellular vaccines against cancer, an approach that has produced limited immune responses and tumor regressions in patients with late-stage disease. Contrastingly, with respect to therapy of organ transplant rejection, we highlight herein how immature/maturation-resistant dendritic cells are emerging as 'negative cellular vaccines', with the ability to induce anergy/apoptosis in alloreactive T cells, while potentially stimulating regulatory T-cell populations. New insights have shed increasing light on dendritic cell immunobiology and the complex processes by which dendritic cell subsets perform both stimulatory and tolerogenic functions. Alloantigen-pulsed host-derived dendritic cells, conditioned with immunosuppressive agents (e.g. rapamycin (RAPA) or dexamethasone) or anti-inflammatory cytokines (e.g. IL-10), are resistance to maturation, and when infused systemically, can promote experimental transplant tolerance, especially when combined with low-dose immunosuppression. Such 'negative' dendritic cell cellular vaccines are proving effective at stimulating/enriching for alloantigen-specific regulatory T cell. Increased understanding of what makes dendritic cells tolerogenic, accompanied by the identification of agents that stably inhibit dendritic cell maturation in the face of proinflammatory stimuli, has given rise to several promising experimental tolerance-inducing protocols. Their translation into clinical testing has the potential to reduce patients' reliance on indefinite, drug-based immunosuppression.
Journal of immunology (Baltimore, Md. : 1950), 2011
IL-33 administration is associated with facilitation of Th2 responses and cardioprotective proper... more IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.
Antigen Processing and Presentation Protocols, 2000
The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains i... more The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains incompletely understood. The important role of foreign peptides in the alloimmune response was recently recognized. We performed a comparative study of the functions of minor antigens Class I, Class II, and CD1 in murine cardiac allograft rejection by investigating the expression of a large panel of immune and inflammatory genes. To investigate the role of MHC Class II and I, our protocol analyzed allograft recipients deficient in MHC Class II and b2 microglobulin (b2-M), a critical component of the Class I heterodimer. We also included CD1 deficient recipients to differentiate effects in the beta2-M deficient strain due to CD1 deficiency versus the combined inactivation of CD1 and Class I. The serum cytokines tumor necrosis factor (TNF)-alpha interleukin (IL)-6, interferon (IFN)-gamma and IL-1beta were evaluated posttransplant by ELISA. The intragraft expression of 55 chemokines, chemokine receptors, and CD markers were measured by ribonuclease protection assay. The data were analyzed through hierarchical clustering dendrograms and self-organizing maps. The analysis indicates that each gene deficiency induces both the upregulation and the downregulation of distinct subsets of genes and that similar kinetics of rejection can be attributed to different molecular mechanisms. The study provides novel insights into the role of classical and non-classical MHC molecules in graft rejection.
ABSTRACT An understanding of one's response to a transplanted organ requires a fundamenta... more ABSTRACT An understanding of one's response to a transplanted organ requires a fundamental understanding of the immune system in general, and its function in the context of physiological homeostasis. The immune system comprises a complex system of tissues, cells, and soluble (humoral) factors that constitute the body's multilayered defenses against disease. It is capable of detecting a wide variety of foreign agents and stages of cellular distress and of distinguishing them from the body's own healthy tissues. Multiple mechanisms mediate the recognition and neutralization of pathogens/foreign material. Evolutionary ancient, non-specific, innate/immediate basic defense mechanisms, such as phagocytosis (mediated by neutrophils, macrophages, and dendritic cells), antimicrobial peptides, and the complement system of proteins, co-exist with more evolutionarily recent elements mediating adaptive or acquired immunity. Adaptive immunity is a property of lymphocytes and, unlike the innate immune system, confers specificity and immunological memory. T lymphocytes mediate and regulate cellular immunity, whereas B cells are involved in production of antibody. The functions of these and other cells involved in host defense are regulated by peptides termed cytokines that are generated in response to challenges to host integrity. Important mechanisms also exist within the immune system to prevent inappropriate attack against self and to maintain self-tolerance.
Herein, we succinctly review mechanisms underlying self-tolerance and the roles of dendritic leuk... more Herein, we succinctly review mechanisms underlying self-tolerance and the roles of dendritic leukocytes (DCs) in T-cell tolerance to self and foreign antigens. We also consider the properties of naturally arising and other populations of regulatory T cells (Treg), together with growing evidence that interplay between DCs and Treg cells can sustain antigen-specific tolerance. B-cell tolerance and the role of hematopoietic cell chimerism in the induction and maintenance of tolerance are also discussed, as is the impact of cosignaling pathway manipulation on tolerance induction. This overview also surveys prospects for technological advances in the monitoring and prediction of tolerance and the application of genomic and proteomic analysis. In addition, we consider potential novel therapeutic targets for promotion of tolerance induction.
Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. Howev... more Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. However, the response rates are limited for current approach that depends on enhancing spontaneous antitumor immune responses. Therefore, increasing tumor immunogenicity by expressing appropriate cytokines should further improve the current immunotherapy. IL-33 is a member of the IL-1 family of cytokines and is released by necrotic epithelial cells or activated innate immune cells and is thus considered a "danger" signal. The role of IL-33 in promoting type 2 immune responses and tissue inflammation has been well established. However, whether IL-33 drives antitumor immune responses is controversial. Our previous work established that IL-33 promoted the function of CD8(+) T cells. In this study, we showed that the expression of IL-33 in two types of cancer cells potently inhibited tumor growth and metastasis. Mechanistically, IL-33 increased numbers and IFN-γ production by CD8(+) T and NK cells in tumor tissues, thereby inducing a tumor microenvironment favoring tumor eradication. Importantly, IL-33 greatly increased tumor Ag-specific CD8(+) T cells. Furthermore, both NK and CD8(+) T cells were required for the antitumor effect of IL-33. Moreover, depletion of regulatory T cells worked synergistically with IL-33 expression for tumor elimination. Our studies established…
Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by even... more Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L(d)/tapasin association can be segregated from reconstitution of folded L(d) surface expression. This finding suggests that peptide acquisition by L(d) is influenced by tapasin functions that are independent of L(d) binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L(d), and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L(d). These data are consistent with a higher overall affinity between L(d) and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin's stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin's electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.
... Drug withdrawal may be required for the management of posttransplant viral infection (ie,Epst... more ... Drug withdrawal may be required for the management of posttransplant viral infection (ie,Epstein-Barr virus, cytomegalovirus, human immunodeficiency virus, hepatitis B or C) or posttransplant lymphoproliferative disease, or may be the result of patient noncompliance or ...
Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents an... more Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents and hold considerable promise as cellular therapeutic agents. Herein, we discuss the ability of regulatory macrophages, regulatory dendritic cells, and myeloid-derived suppressor cells to regulate alloimmunity, their potential as cellular therapeutic agents, and the IS agents that target their function. We consider protocols for the generation of RMC and the selection of donor- or recipient-derived cells for adoptive cell therapy. Additionally, the issues of cell trafficking and antigen (Ag) specificity after RMC transfer are discussed. Improved understanding of the immunobiology of these cells has increased the possibility of moving RMC into the clinic to reduce the burden of current IS agents and to promote Ag-specific tolerance. In the second half of this review, we discuss the influence of established and experimental IS agents on myeloid cell populations. IS agents believed historically to act primarily on T cell activation and proliferation are emerging as important regulators of RMC function. Better insights into the influence of IS agents on RMC will enhance our ability to develop cell therapy protocols to promote the function of these cells. Moreover, novel IS agents may be designed to target RMC in situ to promote Ag-specific immune regulation in transplantation and to usher in a new era of immune modulation exploiting cells of myeloid origin.
Mechanistic target of rapamycin inhibitors (mTORi) have a complex immunoregulatory profile in bot... more Mechanistic target of rapamycin inhibitors (mTORi) have a complex immunoregulatory profile in both animal models and transplant patients. Studies suggest that mTORi act as tolerance-supporting and regulatory T cell (Treg)-promoting immunosuppressants. Yet proinflammatory influences on myeloid dendritic cells have been established. Insight is needed into the impact of mTORi on immune cells. Stallone et al. describe a clinical study identifying a potential immunoregulatory pathway involving plasmacytoid dendritic cells and Tregs in renal transplant patients on mTORi.
This article will describe coordinated analyses of how amino acid substitutions in the HLA class ... more This article will describe coordinated analyses of how amino acid substitutions in the HLA class I antigen binding groove modify chaperone interaction and peptide ligand presentation. By parallel testing of ligand presentation and chaperone interaction with a series of natural HLA-B subtypes, this study has discovered that position 116 of the HLA-B15 class I heavy chain is pivotal in both peptide selection and control of interaction between the assembly complex and the class I heavy chain. Correlated with these qualitative differences in peptide selection and chaperone association are quantitative differences in the expression levels of the HLA molecules at the cell surface. These parallel studies, therefore, demonstrate that particular HLA class I polymorphisms can simultaneously influence ligand presentation and interaction with intracellular chaperones.
Dendritic cells are well known for their potent ability, when fully differentiated or &am... more Dendritic cells are well known for their potent ability, when fully differentiated or 'mature', to stimulate immune responses to antigens they present efficiently to T cells. Mature DC have been used as experimental cellular vaccines against cancer, an approach that has produced limited immune responses and tumor regressions in patients with late-stage disease. Contrastingly, with respect to therapy of organ transplant rejection, we highlight herein how immature/maturation-resistant dendritic cells are emerging as 'negative cellular vaccines', with the ability to induce anergy/apoptosis in alloreactive T cells, while potentially stimulating regulatory T-cell populations. New insights have shed increasing light on dendritic cell immunobiology and the complex processes by which dendritic cell subsets perform both stimulatory and tolerogenic functions. Alloantigen-pulsed host-derived dendritic cells, conditioned with immunosuppressive agents (e.g. rapamycin (RAPA) or dexamethasone) or anti-inflammatory cytokines (e.g. IL-10), are resistance to maturation, and when infused systemically, can promote experimental transplant tolerance, especially when combined with low-dose immunosuppression. Such 'negative' dendritic cell cellular vaccines are proving effective at stimulating/enriching for alloantigen-specific regulatory T cell. Increased understanding of what makes dendritic cells tolerogenic, accompanied by the identification of agents that stably inhibit dendritic cell maturation in the face of proinflammatory stimuli, has given rise to several promising experimental tolerance-inducing protocols. Their translation into clinical testing has the potential to reduce patients' reliance on indefinite, drug-based immunosuppression.
Journal of immunology (Baltimore, Md. : 1950), 2011
IL-33 administration is associated with facilitation of Th2 responses and cardioprotective proper... more IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.
Antigen Processing and Presentation Protocols, 2000
The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains i... more The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains incompletely understood. The important role of foreign peptides in the alloimmune response was recently recognized. We performed a comparative study of the functions of minor antigens Class I, Class II, and CD1 in murine cardiac allograft rejection by investigating the expression of a large panel of immune and inflammatory genes. To investigate the role of MHC Class II and I, our protocol analyzed allograft recipients deficient in MHC Class II and b2 microglobulin (b2-M), a critical component of the Class I heterodimer. We also included CD1 deficient recipients to differentiate effects in the beta2-M deficient strain due to CD1 deficiency versus the combined inactivation of CD1 and Class I. The serum cytokines tumor necrosis factor (TNF)-alpha interleukin (IL)-6, interferon (IFN)-gamma and IL-1beta were evaluated posttransplant by ELISA. The intragraft expression of 55 chemokines, chemokine receptors, and CD markers were measured by ribonuclease protection assay. The data were analyzed through hierarchical clustering dendrograms and self-organizing maps. The analysis indicates that each gene deficiency induces both the upregulation and the downregulation of distinct subsets of genes and that similar kinetics of rejection can be attributed to different molecular mechanisms. The study provides novel insights into the role of classical and non-classical MHC molecules in graft rejection.
ABSTRACT An understanding of one's response to a transplanted organ requires a fundamenta... more ABSTRACT An understanding of one's response to a transplanted organ requires a fundamental understanding of the immune system in general, and its function in the context of physiological homeostasis. The immune system comprises a complex system of tissues, cells, and soluble (humoral) factors that constitute the body's multilayered defenses against disease. It is capable of detecting a wide variety of foreign agents and stages of cellular distress and of distinguishing them from the body's own healthy tissues. Multiple mechanisms mediate the recognition and neutralization of pathogens/foreign material. Evolutionary ancient, non-specific, innate/immediate basic defense mechanisms, such as phagocytosis (mediated by neutrophils, macrophages, and dendritic cells), antimicrobial peptides, and the complement system of proteins, co-exist with more evolutionarily recent elements mediating adaptive or acquired immunity. Adaptive immunity is a property of lymphocytes and, unlike the innate immune system, confers specificity and immunological memory. T lymphocytes mediate and regulate cellular immunity, whereas B cells are involved in production of antibody. The functions of these and other cells involved in host defense are regulated by peptides termed cytokines that are generated in response to challenges to host integrity. Important mechanisms also exist within the immune system to prevent inappropriate attack against self and to maintain self-tolerance.
Herein, we succinctly review mechanisms underlying self-tolerance and the roles of dendritic leuk... more Herein, we succinctly review mechanisms underlying self-tolerance and the roles of dendritic leukocytes (DCs) in T-cell tolerance to self and foreign antigens. We also consider the properties of naturally arising and other populations of regulatory T cells (Treg), together with growing evidence that interplay between DCs and Treg cells can sustain antigen-specific tolerance. B-cell tolerance and the role of hematopoietic cell chimerism in the induction and maintenance of tolerance are also discussed, as is the impact of cosignaling pathway manipulation on tolerance induction. This overview also surveys prospects for technological advances in the monitoring and prediction of tolerance and the application of genomic and proteomic analysis. In addition, we consider potential novel therapeutic targets for promotion of tolerance induction.
Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. Howev... more Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. However, the response rates are limited for current approach that depends on enhancing spontaneous antitumor immune responses. Therefore, increasing tumor immunogenicity by expressing appropriate cytokines should further improve the current immunotherapy. IL-33 is a member of the IL-1 family of cytokines and is released by necrotic epithelial cells or activated innate immune cells and is thus considered a "danger" signal. The role of IL-33 in promoting type 2 immune responses and tissue inflammation has been well established. However, whether IL-33 drives antitumor immune responses is controversial. Our previous work established that IL-33 promoted the function of CD8(+) T cells. In this study, we showed that the expression of IL-33 in two types of cancer cells potently inhibited tumor growth and metastasis. Mechanistically, IL-33 increased numbers and IFN-γ production by CD8(+) T and NK cells in tumor tissues, thereby inducing a tumor microenvironment favoring tumor eradication. Importantly, IL-33 greatly increased tumor Ag-specific CD8(+) T cells. Furthermore, both NK and CD8(+) T cells were required for the antitumor effect of IL-33. Moreover, depletion of regulatory T cells worked synergistically with IL-33 expression for tumor elimination. Our studies established…
Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by even... more Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L(d)/tapasin association can be segregated from reconstitution of folded L(d) surface expression. This finding suggests that peptide acquisition by L(d) is influenced by tapasin functions that are independent of L(d) binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L(d), and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L(d). These data are consistent with a higher overall affinity between L(d) and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin's stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin's electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.
... Drug withdrawal may be required for the management of posttransplant viral infection (ie,Epst... more ... Drug withdrawal may be required for the management of posttransplant viral infection (ie,Epstein-Barr virus, cytomegalovirus, human immunodeficiency virus, hepatitis B or C) or posttransplant lymphoproliferative disease, or may be the result of patient noncompliance or ...
Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents an... more Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents and hold considerable promise as cellular therapeutic agents. Herein, we discuss the ability of regulatory macrophages, regulatory dendritic cells, and myeloid-derived suppressor cells to regulate alloimmunity, their potential as cellular therapeutic agents, and the IS agents that target their function. We consider protocols for the generation of RMC and the selection of donor- or recipient-derived cells for adoptive cell therapy. Additionally, the issues of cell trafficking and antigen (Ag) specificity after RMC transfer are discussed. Improved understanding of the immunobiology of these cells has increased the possibility of moving RMC into the clinic to reduce the burden of current IS agents and to promote Ag-specific tolerance. In the second half of this review, we discuss the influence of established and experimental IS agents on myeloid cell populations. IS agents believed historically to act primarily on T cell activation and proliferation are emerging as important regulators of RMC function. Better insights into the influence of IS agents on RMC will enhance our ability to develop cell therapy protocols to promote the function of these cells. Moreover, novel IS agents may be designed to target RMC in situ to promote Ag-specific immune regulation in transplantation and to usher in a new era of immune modulation exploiting cells of myeloid origin.
Mechanistic target of rapamycin inhibitors (mTORi) have a complex immunoregulatory profile in bot... more Mechanistic target of rapamycin inhibitors (mTORi) have a complex immunoregulatory profile in both animal models and transplant patients. Studies suggest that mTORi act as tolerance-supporting and regulatory T cell (Treg)-promoting immunosuppressants. Yet proinflammatory influences on myeloid dendritic cells have been established. Insight is needed into the impact of mTORi on immune cells. Stallone et al. describe a clinical study identifying a potential immunoregulatory pathway involving plasmacytoid dendritic cells and Tregs in renal transplant patients on mTORi.
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Papers by Heth Turnquist