Bei Liu

Cell surface expression of an endoplasmic reticulum resident heat shock protein gp96 triggers MyD88-dependent systemic autoimmune diseases

Heat shock proteins have been implicated as endogenous activators for dendritic cells (DCs). Without tissue distress or death, these intracellular molecules are inaccessible to surface receptor(s) on DCs, possibly to avoid uncontrolled DC activation and breakdown of immunologic tolerance. We herein addressed this hypothesis in transgenic mice by enforcing cell surface expression of gp96, a ubiquitous heat shock protein of the endoplasmic reticulum. Although a pan-specific promoter is used for transgene expression, neither the expression level nor the tissue distribution of the endogenous gp96 was altered by this maneuver. However, cell surface gp96 induced significant DC activations and spontaneous lupus-like autoimmune diseases, even though the development/functions of lymphocytic compartments were unaltered. Using a bone marrow chimera approach, we further demonstrated that both DC activation and autoimmunity elicited by cell surface gp96 are dependent on the downstream adaptor protein MyD88 for signaling by Toll/IL-1 receptor family. Our study not only established the proinflammatory property of cell surface gp96 in vivo, but also suggested a chronic stimulation of DCs by gp96 as a pathway to initiate spontaneous autoimmune diseases.

TLR4 Up-Regulation at Protein or Gene Level Is Pathogenic for Lupus-Like Autoimmune Disease

TLR4 is the receptor for the Gram-negative bacterial cell wall component LPS. TLR4 signaling is controlled by both positive and negative regulators to balance optimal immune response and potential sepsis. Unchecked TLR4 activation might result in autoimmune diseases, a hypothesis that has not been formally resolved. In this study, we found that TLR4 signaling to LPS can be positively enforced by expressing gp96 on cell surfaces through the chaperone function of, but not the direct signaling by, gp96; TLR4 as well as the commensal flora are essential for the production of anti-dsDNA Ab and the immune complex-mediated glomerulonephritis in transgenic mice that express surface gp96. Moreover, a similar constellation of autoimmunity was evident in mice that encode multiple copies of tlr4 gene. Our study has revealed that increased TLR4 signaling alone without exogenous insult can break immunological tolerance. It provides a strong experimental evidence for TLR4 dysregulation as an etiology of lupus-like renal disease.

Endoplasmic reticulum HSP90b1 (gp96, grp94) optimizes B cell function via chaperoning integrin and TLR but not immunoglobulin

Endoplasmic reticulum (ER) unfolded protein response (UPR) plays pivotal roles in both early B-cell development and plasma cell differentiation. As a major ER chaperone to mediate the UPR and a master chaperone for Toll-like receptors (TLRs), HSP90b1 (grp94, gp96) has long been implicated to facilitate the assembly of immunoglobulin. We hereby critically and comprehensively examine the roles of HSP90b1 in B-cell biology in vivo using B-cell-specific HSP90b1-null mice. We found that knockout B cells developed normally. There were no apparent problems with plasma cell differentiation, Ig assembly, class-switching, and Ig production. Strikingly, although both mutant conventional and innatelike B cells failed to compartmentalize properly due to loss of select but not all integrins, HSP90b1 was required for neither germinal center formation nor memory antibody responses in vivo. The only significant defect associated with HSP90b1 ablation in B cells was an attenuated antibody production in the context of TLR stimulation. Thus, our study has resolved the long-standing question regarding HSP90b1 in B-cell biology: HSP90b1 optimizes the function of B cells by chaperoning TLRs and integrins but not immunoglobulin. This study also has important implications in resolving the controversial roles of TLR in B-cell biology.

Combination of Imatinib Mesylate with Autologous Leukocyte-Derived Heat Shock Protein and Chronic Myelogenous Leukemia

Purpose: To test the feasibility, safety, immunogenicity, and clinical efficacy of an autologous vaccine of leukocyte-derived heat shock protein 70-peptide complexes (Hsp70PC), in conjunction with imatinib mesylate, in patients with chronic myeloid leukemia (CML) in chronic phase. Experimental Design: Patients had cytogenetic or molecular evidence of disease, despite treatment with imatinib mesylate for all except one patient, at the beginning of study. Hsp70PCs were purified from the leukopheresed peripheral blood mononuclear cells and were administered in eight weekly intradermal injections at 50 μg/dose without adjuvant. Clinical responses were assessed by bone marrow analysis before and after vaccinations. An IFN-γ enzyme-linked immunospot assay was used to estimate the effect of treatment on natural killer cells and T cells against CML. Results: Twenty patients were treated. The manufacturing of Hsp70PCs was successful and the administration was safe for all patients. Minimal or no side effects were reported. Clinical responses were seen in 13 of 20 patients as measured by cytogenetic analysis of bone marrow Philadelphia chromosome–positive cells in metaphases and/or, when possible, the level of Bcr/Abl transcript by PCR. Immunologic responses were observed in 9 of 16 patients analyzed, characterized by an increase in the frequency of CML-specific IFN-γ-producing cells and IFN-γ-secreting natural killer cells in the blood. A significant correlation between clinical responses and immunologic responses was observed. Conclusions: Autologous Hsp70PC vaccination is feasible and safe. When combined with imatinib mesylate, it is associated with immunologic and possible clinical responses against CML in chronic phase.

gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis.

Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. The combined role of TLRs and integrin on hematopoiesis remains unclear. gp96 (grp94, HSP90b1) is an endoplasmic reticulum master chaperone for multiple TLRs. We report herein that gp96 is also essential for expression of 14 hematopoietic system-specific integrins. Genetic deletion of gp96 thus enables us to determine the collective roles of gp96, integrins, and TLRs in hematopoiesis. We found that gp96-null hematopoietic stem cells could support long-term myelopoiesis. B- and T-cell development, however, was severely compromised with transitional block from pro-B to pre-B cells and the inability of thymocytes to develop beyond the CD4(-)CD8(-) stage. These defects were cell-intrinsic and could be recapitulated on bone marrow stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not the result of the defect in the assembly of antigen receptor or interleukin-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T- and B-cell development.

The Molecular Chaperone gp96/GRP94 Interacts with Toll-like Receptors and Integrins via Its C-terminal Hydrophobic Domain

Background: gp96 is a molecular chaperone that binds and chaperones TLRs and integrins. But the structural basis of such is unknown. Results: Mutation of a C-terminal loop structure in gp96 abrogates its ability to bind and chaperone both receptors. Conclusion: We have successfully mapped the client-binding domain of gp96. Significance: This study shall facilitate the development of targeted gp96 inhibitors for treatment of inflammation. The structural basis for molecular chaperones to discern misfolded proteins has long been an enigma. As the endoplasmic reticulum paralogue of the cytosolic HSP90, gp96 (GRP94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. However, little is known about its client-binding domain (CBD). Herein, we provide genetic and biochemical evidence to definitively demonstrate that a C-terminal loop structure, formed by residues 652–678, is the critical region of CBD for both TLRs and integrins. Deletion of this region affects neither the intrinsic ATPase activity nor the overall conformation of gp96. However, without it, the chaperoning function of gp96 collapses. We also find a critical Met pair (Met658-Met662) for the folding of integrins but not TLRs. Moreover, we find that the TLR binding to gp96 is also dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. Our study has unveiled surprisingly the exquisite specificity of gp96 in substrate binding and suggests a manipulation of its CBD as an alternative strategy for targeted therapy of a variety of diseases.

Ovarian cancer immunotherapy: opportunities, progresses and challenges.

Due to the low survival rates from invasive ovarian cancer, new effective treatment modalities are urgently needed. Compelling evidence indicates that the immune response against ovarian cancer may play an important role in controlling this disease. We herein summarize multiple immune-based strategies that have been proposed and tested for potential therapeutic benefit against advanced stage ovarian cancer. We will examine the evidence for the premise that an effective therapeutic vaccine against ovarian cancer is useful not only for inducing remission of the disease but also for preventing disease relapse. We will also highlight the questions and challenges in the development of ovarian cancer vaccines, and critically discuss the limitations of some of the existing immunotherapeutic strategies. Finally, we will summarize our own experience on the use of patient-specific tumor-derived heat shock protein-peptide complex for the treatment of advanced ovarian cancer.

Regulatory T cells and Toll-like receptors: what is the missing link?

Toll-like receptors (TLRs) are critical sensors for microbial products and are important in initiating both innate and adaptive immune defenses against pathogens. Emerging evidence suggests that TLRs are also expressed by regulatory T cells (Treg) that constitute an important immune suppressive cellular mechanism to curtail TLR hyperactivity to avoid sepsis and autoimmune diseases. This review brings up to date on the expression of functional TLRs on Treg and the functional impact of TLR activation on Treg biology. We argue that the suppressive function of Treg can be augmented or attenuated depending on the nature of TLR stimulations.

An integrated view of the roles and mechanisms of heat shock protein gp96-peptide complex in eliciting immune response.

Heat shock protein (HSP) gp96, or grp94 is an endoplasmic reticular (ER) paralog of the cytosolic HSP90. Being abundant and non-polymorphic, gp96 plays significant roles in maintaining protein homeostasis in the secretory pathway. This house-keeping role of gp96 has now been overshadowed by the intriguing findings that gp96 modulates both the innate and adaptive components of the immune system. It has been found that, (i) gp96 is one of the major peptide binding proteins in the ER, (ii) gp96 interacts specifically with receptors including CD91 and possibly toll-like receptors (TLRs), on the surface of professional antigen presenting cells (APCs), (iii) interaction with APCs leads to re-presentation of gp96-chaperoned peptides to the major histocompatibility complex (MHC) molecules of APCs, (iv) direct access of gp96 to APCs triggers functional activation of APCs. In this review, we will examine each of these immunological attributes of gp96 critically. As experimentalists, we will also propose specific experiments to examine the argument that gp96, perhaps along with other members of HSP family, is the antigenic carrier for mediating cross priming of antigen-specific T lymphocytes in vertebrates.

TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein gp96 Potentiates Suppressive Function of Regulatory T Cells

As one of the main mediators of the endoplasmic reticulum unfolded protein response, heat shock protein gp96 is also an obligate chaperone for multiple TLRs including TLR4. We demonstrated recently that enforced cell surface expression of gp96 in a transgenic (Tg) mouse (96tm-Tg) conferred hyperresponsiveness to LPS and induced TLR4-dependent lupus-like autoimmune diseases. In this study, we investigated the function of CD4+CD25+ Foxp3+ regulatory T cells (Treg) in these mice in light of the important roles of Treg in the maintenance of peripheral tolerance against self-Ag as well as the increasing appreciation of TLR signaling on the regulation of Treg. We found that the development of Treg was not impaired in 96tm-Tg mice. Contrary to the prediction of dampened Treg activity, we discovered that the suppressive functions of Treg were increased in 96tm-Tg mice. Inactivation of Treg during the neonatal stage of life exacerbated not only organ-specific diseases but also systemic autoimmune diseases. By crossing 96tm-Tg mice into the TLR4 null background, we demonstrated the critical roles of TLR4 in the amplification of Treg suppressive function. These findings illustrate that gp96 plays dual roles in regulating immune responses by augmenting proinflammatory responses and inducing Treg function, both of which are dependent on its ability to chaperone TLR4. Our study provides strong support to the notion of compensatory Treg activation by TLR ligation to dampen inflammation and autoimmune diseases.