IL-18-secreting multi-antigen targeting CAR T-cells eliminate antigen-low myeloma in an immunocompetent mouse model.

Multiple myeloma is a plasma cell malignancy that is currently incurable with conventional therapies. Following the success of CD19-targeted chimeric antigen receptor (CAR) T-cells in leukemia and lymphoma, CAR T-cells targeting B-cell maturation antigen (BCMA) more recently demonstrated impressive activity in relapsed and refractory myeloma patients. However, BCMA-directed therapy can fail due to low expression of BCMA on myeloma cells, suggesting that novel approaches to better address antigen-low disease may improve patient outcomes. We hypothesized that engineered secretion of the pro-inflammatory cytokine interleukin-18 (IL-18) and multi-antigen targeting could improve CAR T-cell activity against BCMA-low myeloma. In a syngeneic murine model of myeloma, CAR T-cells targeting the myeloma-associated antigens BCMA and B-cell activating factor (BAFF-R) failed to eliminate myeloma when these antigens were weakly expressed, whereas IL-18-secreting CAR T-cells targeting these antigens promoted myeloma clearance. IL-18-secreting CAR T-cells developed an effector-like T-cell phenotype, promoted interferon-gamma production, reprogrammed the myeloma bone marrow microenvironment through type I/II interferon signaling, and activated macrophages to mediate anti-myeloma activity. Simultaneous targeting of weakly expressed BCMA and BAFF-R with dual-CAR T-cells enhanced T-cell:target cell avidity, increased overall CAR signal strength, and stimulated anti-myeloma activity. Dual-antigen targeting augmented CAR T-cell secretion of engineered IL-18 and facilitated elimination of larger myeloma burdens in vivo. Our results demonstrate that combination of engineered IL-18 secretion and multi-antigen targeting can eliminate myeloma with weak antigen expression through distinct mechanisms.

Tracking the cellular immune response from early premalignancy to active multiple myeloma in a new model.

To calibrate a murine model to study premalignant to malignant multiple myeloma, mice were inoculated with different amounts of myeloma cells, and changes in the immune profile were tracked for over 200 days. The model highlights the development of T-cell exhaustion and suppressor before the appearance of clinical symptoms.

Id-neoantigen vaccine induces therapeutic CD8+ T cells against multiple myeloma: H chain-loss escapees cause FLC MM.

BACKGROUND: Multiple myeloma (MM) cancers originate from plasma cells that have passed through the germinal center reaction where somatic hypermutation of Ig V regions takes place. Myeloma protein V regions often express many mutations and are thus a rich source of neoantigens (traditionally called idiotopes (Id)). Therefore, these are highly tumor-specific and excellent targets for immunotherapy. METHODS: We have developed a DNA Id vaccine which as translated protein targets conventional dendritic cells (cDC) for CCL3-mediated delivery of myeloma protein V regions in a single-chain fragment variable (scFv) format. Vaccine efficacy was studied in the mouse MM model, mineral oil-induced plasmacytoma 315.BM. RESULTS: The Id vaccine protected mice against a challenge with MM cells. Moreover, the vaccine had a therapeutic effect. However, in some of the vaccinated mice, MM cells not producing H chains escaped rejection, resulting in free light chain (FLC) MM. Depletion of CD8+ T cells abrogated vaccine efficacy, and protection was observed to be dependent on cDC1s, using Batf3-/- mice. Modifications of scFv in the vaccine demonstrated that CD8+ T cells were specific for two mutated VH sequences. CONCLUSIONS: VH neoantigen-specific CD8+ T cells elicited by CCL3-containing Id vaccines had a therapeutic effect against MM in a mouse model. MM cells could escape rejection by losing expression of the H chain, thus giving rise to FLC MM.

Preclinical Evaluation of a 64Cu-Based Theranostic Approach in a Murine Model of Multiple Myeloma.

Although the concept of theranostics is neither new nor exclusive to nuclear medicine, it is a particularly promising approach for the future of nuclear oncology. This approach is based on the use of molecules targeting specific biomarkers in the tumour or its microenvironment, associated with optimal radionuclides which, depending on their emission properties, allow the combination of diagnosis by molecular imaging and targeted radionuclide therapy (TRT). Copper-64 has suitable decay properties (both ß+ and ß- decays) for PET imaging and potentially for TRT, making it both an imaging and therapy agent. We developed and evaluated a theranostic approach using a copper-64 radiolabelled anti-CD138 antibody, [64Cu]Cu-TE1PA-9E7.4 in a MOPC315.BM mouse model of multiple myeloma. PET imaging using [64Cu]Cu-TE1PA-9E7.4 allows for high-resolution PET images. Dosimetric estimation from ex vivo biodistribution data revealed acceptable delivered doses to healthy organs and tissues, and a very encouraging tumour absorbed dose for TRT applications. Therapeutic efficacy resulting in delayed tumour growth and increased survival without inducing major or irreversible toxicity has been observed with 2 doses of 35 MBq administered at a 2-week interval. Repeated injections of [64Cu]Cu-TE1PA-9E7.4 are safe and can be effective for TRT application in this syngeneic preclinical model of MM.

EMD originates from hyaluronan-induced homophilic interactions of CD44 variant-expressing MM cells under shear stress.

Extramedullary disease (EMD) is known to be associated with chemoresistance and poor prognosis in multiple myeloma (MM); however, the mechanisms of its development are not fully understood. Elucidating the mechanism of EMD development and its therapeutic targeting would greatly contribute to further improvement of treatment outcome in patients with MM. Here, we show that bone marrow stroma cell-derived hyaluronan (HA) elicits homophilic interactions of MM cells by binding to surface CD44, especially long-stretch variants, under physiological shear stress and generates cell clusters that might develop into EMD. We recapitulated the development of EMD via administration of HA in a syngeneic murine MM model in a CD44-dependent manner. HA-induced MM cell clusters exhibited the specific resistance to proteasome inhibitors (PIs) in vitro and in murine models via γ-secretase-mediated cleavage of the intracellular domains of CD44, which in turn transactivated PI resistance-inducible genes. Treatment of HA-injected mice with anti-CD44 antibody or γ-secretase inhibitors readily suppressed the development of EMD from transplanted MM cells and significantly prolonged the survival of recipients by overcoming PI resistance. The HA-CD44 axis represents a novel pathway to trigger EMD development and could be a target of the prediction, prevention, and treatment of EMD in patients with MM.

VSIG4-expressing tumor-associated macrophages impair anti-tumor immunity.

VSIG4, a newly identified co-inhibitory molecule belonging to the B7-related family, is exclusively expressed on tissue-resident macrophages and is involved in the suppression of T cell proliferation and cytokine production. We sought to characterize the role of VSIG4 in anti-tumor immunity in the tumor microenvironment, focusing on VSIG4-expressing tumor-associated macrophages (TAMs). We found that VSIG4-expressing TAMs negatively regulated antigen-specific T cell proliferation and cytokine production through direct inhibition via cell cycle arrest, but not apoptosis, as well as through their arginase 1 activity. Furthermore, VSIG4-expressing TAMs suppress tumor-specific CD8+ T cell cytotoxicity. Therefore, our results suggest that VSIG4-expressing TAMs could be a negative cellular regulator of anti-tumor immunity in the tumor microenvironment.

Multiple Myeloma-Derived Extracellular Vesicles Modulate the Bone Marrow Immune Microenvironment.

Multiple myeloma (MM), the third most frequent hematological cancer worldwide, is characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM). One of the hallmarks of MM is a permissive BM microenvironment. Increasing evidence suggests that cell-to-cell communication between myeloma and immune cells via tumor cell-derived extracellular vesicles (EV) plays a key role in the pathogenesis of MM. Hence, we aimed to explore BM immune alterations induced by MM-derived EV. For this, we inoculated immunocompetent BALB/cByJ mice with a myeloma cell line, MOPC315.BM, inducing a MM phenotype. Upon tumor establishment, characterization of the BM microenvironment revealed the expression of both activation and suppressive markers by lymphocytes, such as granzyme B and PD-1, respectively. In addition, conditioning of the animals with MOPC315.BM-derived EV, before transplantation of the MOPC315.BM tumor cells, did not anticipate the disease phenotype. However, it induced features of suppression in the BM milieu, such as an increase in PD-1 expression by CD4+ T cells. Overall, our findings reveal the involvement of MOPC315.BM-derived EV protein content as promoters of immune niche remodeling, strengthening the importance of assessing the mechanisms by which MM may impact the immune microenvironment.

Antigen bivalency of antigen-presenting cell-targeted vaccines increases B cell responses.

Antibodies are important for vaccine efficacy. Targeting antigens to antigen-presenting cells (APCs) increases antibody levels. Here, we explore the role of antigen valency in MHC class II (MHCII)-targeted vaccines delivered as DNA. We design heterodimeric proteins that carry either two identical (bivalent vaccines), or two different antigens (monovalent vaccines). Bivalent vaccines with two identical influenza hemagglutinins (HA) elicit higher amounts of anti-HA antibodies in mice than monovalent versions with two different HAs. Bivalent vaccines increase the levels of germinal center (GC) B cells and long-lived plasma cells. Only HA-bivalent vaccines completely protect mice against challenge with homologous influenza virus. Similar results are obtained with other antigens by targeting CD11c and Xcr1 on dendritic cells (DCs) or when administering the vaccine as protein with adjuvant. Bivalency probably increases B cell responses by cross-linking BCRs in readily observable DC-B cell synapses. These results are important for generating potent APC-targeted vaccines.

Neoantigen vaccine-induced CD4 T cells confer protective immunity in a mouse model of multiple myeloma through activation of CD8 T cells against non-vaccine, tumor-associated antigens.

BACKGROUND: Cancer-associated neoantigens (neoAg) derived from tumor genomic sequencing and predictive algorithms for mutated peptides are a promising basis for therapeutic vaccines under investigation. Although these are generally designed to bind major histocompatibility complex class I and induce CD8 cytolytic T lymphocyte (CTL) activity, results from preclinical and clinical studies demonstrate that the majority of neoAg vaccines efficiently induce CD4 T helper (Th) responses but not CTL. Despite this, these vaccines have demonstrated clinical efficacy. Therefore, understanding the mechanisms of CD4 + T cell-mediated tumor protection is critical to optimizing this immunotherapeutic strategy. METHODS: We investigated this phenomenon in the mineral oil-induced plasmacytoma (MOPC).315.BM (MOPC315) mouse model of multiple myeloma, a malignancy of plasma cells. MOPC315 cells express in their lambda chain a unique tumor-specific neoAg, an idiotypic (Id) peptide. We generated a vaccine formulated with this Id peptide fused to a heat shock protein HSC70 binding (HSB) motif co-delivered with poly (I:C). The immunogenicity of the Id-vaccine was measured in splenocytes by ELISpot. Mice were challenged with MOPC315 cells and antitumor immunity was assessed by co-incubating splenocytes and bone marrow mononuclear cells derived from vaccinated mice and controls, with the Id antigen and irradiated MOPC315 cells. The frequency of activated CD4 and CD8 T cells and their phenotype were characterized by flow cytometry. RESULTS: Id-vaccine efficiently induced antigen-specific CD4 Th activity and antitumor immunity, protecting mice from MOPC315 tumor growth. CD4 cytolytic activity was not detected under these conditions. Polyfunctional CD8 T cells homed to the bone marrow microenvironment of protected mice and preferentially expanded only when restimulated ex vivo with both Id peptide and MOPC315 cells. Protective activity was abrogated by depletion of either CD4 or CD8 lymphocytes. CONCLUSION: These results demonstrate that Id-HSB +poly (I:C) vaccine protects against MOPC315 growth by priming Id-specific CD4 Th cells that confer protection against tumor but are not directly cytotoxic. These data indicate that activation of CD8 CTL against MOPC315-associated antigens not present in the vaccine is one of the major mechanisms of tumor immunity.

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named "Vaccibody", has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity.

Humoral immunity relies on the efficient differentiation of memory B cells (MBCs) into antibody-secreting cells (ASCs). T helper (Th) signals upregulate B cell receptor (BCR) signaling by potentiating Src family kinases through increasing CD45 phosphatase activity (CD45 PA). In this study, we show that high CD45 PA in MBCs enhances BCR signaling and is essential for their effective ASC differentiation. Mechanistically, Th signals upregulate CD45 PA through intensifying the surface binding of a CD45 ligand, Galectin-1. CD45 PA works as a sensor of T cell help and defines high-affinity germinal center (GC) plasma cell (PC) precursors characterized by IRF4 expression in vivo. Increasing T cell help in vitro results in an incremental CD45 PA increase and enhances ASC differentiation by facilitating effective induction of the transcription factors IRF4 and BLIMP1. This study connects Th signals with BCR signaling through Galectin-1-dependent regulation of CD45 PA and provides a mechanism for efficient ASC differentiation of MBCs.

Prevention of Bone Destruction by Mechanical Loading Is Not Enhanced by the Bruton's Tyrosine Kinase Inhibitor CC-292 in Myeloma Bone Disease.

Limiting bone resorption and regenerating bone tissue are treatment goals in myeloma bone disease (MMBD). Physical stimuli such as mechanical loading prevent bone destruction and enhance bone mass in the MOPC315.BM.Luc model of MMBD. It is unknown whether treatment with the Bruton's tyrosine kinase inhibitor CC-292 (spebrutinib), which regulates osteoclast differentiation and function, augments the anabolic effect of mechanical loading. CC-292 was administered alone and in combination with axial compressive tibial loading in the MOPC315.BM.Luc model for three weeks. However, neither CC-292 alone nor its use in combination with mechanical loading was more effective in reducing osteolytic bone disease or rescuing bone mass than mechanical stimuli alone, as evidenced by microcomputed tomography (microCT) and histomorphometric analysis. Further studies are needed to investigate novel anti-myeloma and anti-resorptive strategies in combination with physical stimuli to improve treatment of MMBD.

ARHGAP45 controls naïve T- and B-cell entry into lymph nodes and T-cell progenitor thymus seeding.

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans-endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase-activating proteins (GAPs). T and B cells express several RHO-GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time-lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T-cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T-cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T-cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.

Mechanical loading prevents bone destruction and exerts anti-tumor effects in the MOPC315.BM.Luc model of myeloma bone disease.

Bone continually adapts to changing external loading conditions via (re)modeling (modeling and remodeling) processes. While physical activity is known to beneficially enhance bone mass in healthy individuals, little is known in how physical stimuli affect osteolytic bone destruction in patients suffering from multiple myeloma bone disease. Multiple myeloma (MM) is caused by malignant plasma cells in the bone marrow, shifting the balance in bone remodeling towards massive resorption. We hypothesized that in vivo tibial mechanical loading has anabolic effects in mice with locally injected MOPC315.BM.Luc cells. Conventional microCT analysis revealed enhanced cortical bone mass and microstructure in loaded compared to nonloaded mice. State-of-the-art time-lapse microCT based image analysis demonstrated bone (re)modeling processes at the endosteal and periosteal surfaces as the underlying causes of increased bone mass. Loading prevented the progression and development of osteolytic destruction. Physical stimuli also diminished local MM cell growth and dissemination evidenced by quantification of MM cell-specific immunoglobulin A levels in the serum of mice and by bioluminescence analysis. These data indicate that mechanical loading not only rescues the bone phenotype, but also exerts cell-extrinsic anti-myeloma effects in the MOPC315.BM.Luc model. In conclusion, the use of physical stimuli should be further investigated as an anabolic treatment for osteolytic bone destruction in patients with MM.

An Early Myeloma Bone Disease Model in Skeletally Mature Mice as a Platform for Biomaterial Characterization of the Extracellular Matrix.

Multiple myeloma (MM) bone disease is characterized by osteolytic bone tissue destruction resulting in bone pain, fractures, vertebral collapse, and spinal cord compression in patients. Upon initial diagnosis of MM, almost 80% of patients suffer from bone disease. Earlier diagnosis and intervention in MM bone disease would potentially improve treatment outcome and patient survival. New preclinical models are needed for developing novel diagnostic markers of bone structural changes as early as possible in the disease course. Here, we report a proof-of-concept, syngeneic, intrafemoral MOPC315.BM MM murine model in skeletally mature BALB/c mice for detection and characterization of very early changes in the extracellular matrix (ECM) of MM-injected animals. Bioluminescence imaging (BLI) in vivo confirmed myeloma engraftment in 100% of the animals with high osteoclast activity within 21 days after tumor cell inoculation. Early signs of aggressive bone turnover were observed on the outer bone surfaces by high-resolution microcomputed tomography (microCT). Synchrotron phase contrast-enhanced microcomputer tomography (PCE-CT) revealed very local microarchitecture differences highlighting numerous active sites of erosion and new bone at the micrometer scale. Correlative backscattered electron imaging (BSE) and confocal laser scanning microscopy allowed direct comparison of mineralized and nonmineralized matrix changes in the cortical bone. The osteocyte lacunar-canalicular network (OLCN) architecture was disorganized, and irregular-shaped osteocyte lacunae were observed in MM-injected bones after 21 days. Our model provides a potential platform to further evaluate pathological MM bone lesion development at the micro- and ultrastructural levels. These promising results make it possible to combine material science and pharmacological investigations that may improve early detection and treatment of MM bone disease.

CD4+ T-cell killing of multiple myeloma cells is mediated by resident bone marrow macrophages.

CD4+ T cells may induce potent antitumor immune responses through interaction with antigen-presenting cells within the tumor microenvironment. Using a murine model of multiple myeloma, we demonstrated that adoptive transfer of idiotype-specific CD4+ T cells may elicit curative responses against established multifocal myeloma in bone marrow. This finding indicates that the myeloma bone marrow niche contains antigen-presenting cells that may be rendered tumoricidal. Given the complexity of the bone marrow microenvironment, the mechanistic basis of such immunotherapeutic responses is not known. Through a functional characterization of antitumor CD4+ T-cell responses within the bone marrow microenvironment, we found that killing of myeloma cells is orchestrated by a population of bone marrow-resident CD11b+F4/80+MHC-IIHigh macrophages that have taken up and present secreted myeloma protein. The present results demonstrate the potential of resident macrophages as powerful mediators of tumor killing within the bone marrow and provide a basis for novel therapeutic strategies against multiple myeloma and other malignancies that affect the bone marrow.

A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza.

Immunogenicity of DNA vaccines can be increased by constructing the DNA in such a way that it encodes secreted homodimeric fusion proteins that target antigen-presenting cells (APCs). In this study, we have developed novel APC-targeting vaccine molecules with an increased flexibility due to introduction of a heterodimerization motif. The heterodimeric proteins permit four different fusions within a single molecule, thus allowing expression of two different APC-targeting moieties and two different antigens. Two types of heterodimeric fusion proteins were developed that employed either the ACID/BASE or the Barnase/Barstar motifs, respectively. The ACID/BASE heterodimeric vaccines conferred protection against challenges with either influenza virus or tumor cells in separate preclinical models. The ACID/BASE motif was flexible since a large number of different targeting moieties and antigens could be introduced with maintenance of specificity, antigenicity, and secretion. APC-targeting ACID/BASE vaccines expressing two different antigens induced antibody and T cell responses against either of the two antigens. Heterodimeric ACID/BASE DNA vaccines were of approximately the same potency as previously reported homodimeric DNA vaccines. The flexibility and potency of the ACID/BASE format suggest that it could be a useful platform for DNA vaccines that encode APC-targeting fusion proteins.

PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGFß Inhibitors.

PURPOSE: Immune-checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma. EXPERIMENTAL DESIGN: Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed multiple myeloma patients. We examined the expression of immune-checkpoint receptors in BM CD8+ T cells and their functional restoration by ex vivo treatment with anti-PD-1 and TGFß inhibitors. RESULTS: We confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of multiple myeloma patients. PD-1-expressing CD8+ T cells from the BM of multiple myeloma patients coexpressed other checkpoint inhibitory receptors and exhibited a terminally differentiated phenotype. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. BM CD8+ T cells from multiple myeloma patients exhibited reduced proliferation and cytokine production upon T-cell receptor stimulation. However, anti-PD-1 did not increase the proliferation of BM CD8+ T cells from multiple myeloma patients, indicating that T-cell exhaustion in multiple myeloma is hardly reversed by PD-1 blockade alone. Intriguingly, anti-PD-1 significantly increased the proliferation of BM CD8+ T cells from multiple myeloma patients in the presence of inhibitors of TGFß, which was overexpressed by myeloma cells. CONCLUSIONS: Our findings indicate that combined blockade of PD-1 and TGFß may be useful for the treatment of multiple myeloma.

Functional design of pH-responsive folate-targeted polymer-coated gold nanoparticles for drug delivery and in vivo therapy in breast cancer.

BACKGROUND: Curcumin has been widely used owing to its various medicinal properties including antitumor effects. However, its clinical application is limited by its instability, poor solubility and low bioavailability. Folic acid (FA)-functionalized nanoformulations may enhance the sustained release of an anticancer drug (curcumin) by tumor-specific targeting to improve therapeutic benefit. This study aims to design a nanoconjugate (NC) comprised of folate-curcumin-loaded gold-polyvinylpyrrolidone nanoparticles (FA-CurAu-PVP NPs) for targeted delivery in breast cancer model systems. METHODS: We developed curcumin-loaded FA-functionalized Au-PVP NCs by layer-by-layer assembly. The folic acid-curcumin Au-PVP NCs (FA-CurAu-PVP NCs) were characterized by ultraviolet-visible spectra, Fourier transform infrared spectroscopy, X-ray powder diffraction and thermogravimetric analysis. In vitro anticancer and antimigratory effects of NCs were examined by performing MTT and wound migration assays. The in vivo antitumor efficacy of NCs was investigated using a preclinical breast cancer orthotopic mouse model. RESULTS: Curcumin (40 µg/mL) was loaded along with conjugation of folate onto Au-PVP NPs to form FA-CurAu-PVP NCs. The size and charge of the NCs were increased gradually through layer-by-layer assembly and showed 80% release of curcumin at acidic pH. The NC did not show aggregation when incubated with human serum and mimicked an intrinsic peroxidase-like property in the presence of 3,3',5,5'-tetramethylbenzidine substrate. The MTT data using these NCs showed efficient anticancer activity at lower doses in estrogen/progesterone receptor (ER/PR)-negative cells compared with ER/PR-positive cells. Furthermore, the NCs did not show cytotoxicity at the investigated concentration in human breast epithelial and mouse fibroblast cell lines. They showed inhibitory effects on cell migration and high antitumor efficacy in in vivo analysis. CONCLUSION: These results suggest that folate-based tumor targeting using CurAu-PVP NCs is a promising approach for tumor-specific therapy of breast cancer without harming normal cells.

B cell receptor ligation induces display of V-region peptides on MHC class II molecules to T cells.

The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.