Ning Fu

Blockade of Programmed Death-1 Pathway Rescues the Effector Function of Tumor-Infiltrating T Cells and Enhances the Antitumor Efficacy of Lentivector Immunization

Despite intensive effort, the antitumor efficacy of tumor vaccines remains limited in treating established tumors regardless of the potent systemic tumor-specific immune response and the increases of tumor infiltration of T effector cells. In the current study, we demonstrated that although lentivector (lv) immunization markedly increased Ag-dependent tumor infiltration of CD8 and CD4 T cells and generated Ag-specific antitumor effect, it simultaneously increased the absolute number of myeloid-derived suppressor cells and regulatory T cells in the tumor lesions. In addition, lv immunization induced expression of programmed death-ligand 1 in the tumor lesions. Furthermore, the tumor-infiltrating CD8 T cells expressed high levels of programmed death-1 and were partially dysfunctional, producing lower amounts of effector cytokines and possessing a reduced cytotoxicity. Together, these immune-suppression mechanisms in the tumor microenvironment pose a major obstacle to effective tumor immunotherapy and may explain the limited antitumor efficacy of lv immunization. The loss of effector function in the tumor microenvironment is reversible, and the effector function of CD8 T cells in the tumor could be partially rescued by blocking programmed death-1 and programmed death-ligand 1 pathway in vitro and in vivo, resulting in enhanced antitumor efficacy of lv immunization. These data suggest that immunization alone may exacerbate immune suppression in the tumor lesions and that methods to improve the tumor microenvironment and to rescue the effector functions of tumor-infiltrating T cells should be incorporated into immunization strategies to achieve enhanced antitumor efficacy.

Lentivector Immunization Stimulates Potent CD8 T Cell Responses against Melanoma Self-Antigen Tyrosinase-Related Protein 1 and Generates Antitumor Immunity in Mice

Recombinant lentivector immunization has been demonstrated to induce potent CD8 T cell responses in vivo. In this study, we investigated whether lentivector delivering a self/tumor Ag, tyrosinase related protein 1 (TRP1), could stimulate effective antitumor T cell responses. We found that immunization with lentivector expressing mutated TRP1 Ag elicited potent CD8 T cell responses against multiple TRP1 epitopes. Importantly, the activated CD8 T cells effectively recognize wild-type TRP1 epitopes. At peak times, as many as 10% of CD8 T cells were effector cells against TRP1 Ag. These cells killed wild-type TRP1 peptide-pulsed target cells in vivo and produced IFN-γ after ex vivo stimulation. The CD8 T cell responses were long-lasting (3–4 wk). Immunized mice were protected from B16 tumor cell challenge. In a therapeutic setting, lentivector immunization induced potent CD8 T cell responses in tumor bearing mice. The number of infiltrating T cells and the ratio of CD8/CD4 were dramatically increased in the tumors of immunized mice. The tumor-infiltrating CD8 T cells were functional and produced IFN-γ. The potent CD8 T cell responses stimulated by lentivector immunization eliminated small 3-day s.c. B16 tumors and strongly inhibited the growth of more established 5-day tumors. These studies demonstrate that genetic immunization with lentivector expressing mutated self/tumor Ag can generate potent CD8 T cell immune responses and antitumor immunity that prevent and inhibit B16 tumor growth, suggesting that lentivector immunization has the potential for tumor immunotherapy and immune prevention.

Local Administration of TLR Ligands Rescues the Function of Tumor-Infiltrating CD8 T Cells and Enhances the Antitumor Effect of Lentivector Immunization

Cancer vaccines, to date, have shown limited effect to control the growth of established tumors due largely to effector failure of the antitumor immune responses. Tumor lesion is characterized as chronic indolent inflammation in which the effector function of tumor-infiltrating lymphocytes (TILs) is severely impaired. In this study, we investigated whether the effector function of CD8 TILs could be rescued by converting the chronic inflammation milieu to acute inflammation within tumors. We found that injection of TLR3/9 ligands (polyI:C/CpG) into a tumor during the effector phase of lentivector (lv) immunization effectively rescued the function of lv-activated CD8 TILs and decreased the percentage of T regulatory within the tumor, resulting in a marked improvement in the antitumor efficacy of lv immunization. Mechanistically, rescue of the effector function of CD8 TILs by TLR3/9 ligands is most likely dependent on production, within a tumor, of type-1 IFN that can mature and activate tumor-infiltrating dendritic cells. The effector function of CD8 TILs could not be rescued in mice lacking intact type I IFN signaling. These findings have important implications for tumor immunotherapy, suggesting that type I IFN-mediated activation of tumor-infiltrating dendritic cells within a tumor will most likely restore/enhance the effector function of CD8 TILs and thus improve the antitumor efficacy of current cancer vaccines.

Lentivector Prime and Vaccinia Virus Vector Boost Generate High-Quality CD8 Memory T Cells and Prevent Autochthonous Mouse Melanoma

Most cancer vaccines, to date, fail to control established tumors. However, their application in preventing tumors is another question that is understudied. In the current study, we investigated the CD8 memory T cell responses of lentivector (lv) immunization and its potential to prevent melanoma using both transplantable B16 tumor and autochthonous melanoma models. We found that lv-expressing xenogenic human gp100 could induce potent CD8 responses that cross-react with mouse gp100. Importantly, the lv-primed CD8 response consisted of a high number of memory precursors and could be further increased by recombinant vaccinia virus vector (vv) boost, resulting in enhanced CD8 memory response. These long-lasting CD8 memory T cells played a critical role in immune surveillance and could rapidly respond and expand after sensing B16 tumor cells to prevent tumor establishment. Although CD8 response plays a dominant role after lv immunization, both CD4 and CD8 T cells are responsible for the immune prevention. In addition, we surprisingly found that CD4 help was not only critical for generating primary CD8 responses, but also important for secondary CD8 responses of vv boost. CD4 depletion prior to lv prime or prior to vv boost substantially reduced the magnitude of secondary CD8 effector and memory responses, and severely compromised the effect of cancer immune prevention. More importantly, the CD8 memory response from lv-vv prime-boost immunization could effectively prevent autochthonous melanoma in tumor-prone transgenic mice, providing a strong evidence that lv-vv prime-boost strategy is an effective approach for cancer immune prevention.

Peptide mimics of peptidoglycan are vaccine candidates and protect mice from infection with Staphylococcus aureus

Staphylococcus aureus drug resistance to antibiotics is a serious situation that has drawn greater attention to immunotherapy and prophylaxis. Peptidoglycan (PGN) is a common and conserved component of the cell wall of Gram-positive bacteria such as S. aureus. However, PGN, as a thymus-independent antigen, cannot be considered a vaccine candidate because of its very weak immunogenicity. In this study we have attempted to enhance the immunogenicity of PGN by identifying a PGN peptide mimic sequence that would act as a thymus-dependent antigen. Several peptide sequences were obtained from a phage display peptide library using a mAb against S. aureus PGN, and a 12-mer linear single peptide (Sp-31) and a four-branch multiple antigen peptide (MAP) (MAP-P31) were synthesized. Both Sp-31 and MAP-P31 were shown to bind directly to anti-PGN mAb and a polyclonal antibody against S. aureus. These peptides could also inhibit the binding of PGN to a mAb against PGN. Furthermore, MAP-P31 was able to provoke an effective secondary antibody response in mice to PGN and to cell-wall fragments isolated from S. aureus, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa by sonication. In addition, the MAP-P31 antiserum showed a potent bactericidal or bacteriostatic activity against S. aureus in the presence and absence of complement in vitro. Importantly, immunization with MAP-P31 significantly prolonged the survival and enhanced bacterial clearance in BALB/c mice challenged with live S. aureus. In addition, the serum IgG-type antibodies against PGN persisted in mice, demonstrating that MAP-P31, as a peptide mimicking epitopes on PGN, provokes an effective secondary or memory antibody response, which can only be induced by a thymus-dependent antigen and which protects against infection with S. aureus. These results suggest that MAP-31 may be a novel vaccine candidate against S. aureus.

Macrophages are the dominant effector cells responsible for IgG-mediated passive systemic anaphylaxis challenged by natural protein antigen in BALB/c and C57BL/6 mice

IgG-induced passive systemic anaphylaxis (PSA), a serious adverse effect of passive immune therapy using therapeutic monoclonal antibodies, has been greatly emphasized. However, controversy exists regarding the type of effector cells involved in IgG-induced anaphylaxis as a result of the induction of PSA by different IgG subtypes or the use of mice with varying genetic backgrounds. To clarify the effector cells for PSA, the PSA model with serious hypothermia was established by IgG monoclonal antibody (mAb) against natural protein or complete antigen, not hapten conjugate, in BALB/c and C57BL/6 mice. The results indicated that PSA could be remarkably inhibited by the depletion of macrophages but not by the depletion of whole leukocytes, basophils, neutrophils or monocytes. We further confirmed that macrophages are indispensable for the PSA induced by all six IgG-natural antigen complexes in both strains of mice. Additionally, platelet-activating factor (PAF) was found to be the major effector mediator for IgG-induced anaphylaxis. Moreover, gene knock-out of the third component of complement (C3) did not affect PSA-related hypothermia in C57BL/6 mice. These results indicate that macrophages and PAF act as dominant effector cells and mediator molecules, respectively, and are indispensable components in the induction of IgG-mediated PSA induced by IgG mAb and natural protein antigen. Based on the above results, we hypothesize that inconsistencies in effector cells for PSA may be associated with different features of the mAb-antigen system that might affect the magnitude of FcγRs cross-linking on effector cells.

Characteristics of anaphylaxis‐inducing IgG immune complexes triggering murine passive systemic anaphylaxis

With the broad and increasing application of therapeutic monoclonal antibodies (mAbs) in clinical settings, IgG‐induced allergic reactions, including passive systemic anaphylaxis (PSA), have attracted significant attention. However, it is not clear which types of IgG mAb–antigen complexes or IgG aggregates formed by antigen binding can trigger PSA, as not all immune complexes (ICs) are capable of triggering PSA. Here, we characterise mAb–antigen complexes capable of inducing murine PSA to evaluate and predict which ICs are able to induce PSA.

Development of a fluorescence immunochromatographic assay for the detection of zeta globin in the blood of (−−SEA) α-thalassemia carriers

Southeast Asian deletion (--(SEA)) α-thalassemia is an inherited monogenic disorder of human hemoglobin, and embryonic globin ζ (hemoglobin ζ, zeta globin chain or Hb zeta chain) has been shown to be a marker that can be used for the identification of carriers of the (--(SEA)) α-thalassemia deletion. In this work, a fluorescence immunochromatographic assay (FL-ICA) was established to detect the zeta globin chain in the hemolysates of carriers of the (--(SEA)) α-thalassemia deletion. This assay can be completed within 10min using a simple UV detector and does not suffer from interference from the red background color of the hemolysate. A total of 314 blood samples were tested by FL-ICA and ELISA. The results of these assays were confirmed by PCR, the standard technique for genetic disease testing. The sensitivity and specificity of this novel FL-ICA were 100% and 98.0%, respectively; the corresponding values for the ELISA performed simultaneously were 100% and 99.2%, respectively. In conclusion, a new FL-ICA-a simple, fast, convenient, low-cost method-was developed that may be useful in both high-throughput screening and individual detection of the (--(SEA)) α-thalassemia deletion in carriers. Additionally, this qualitative FL-ICA may enlighten the development of a new systems for analysis of other target molecules using whole-blood samples.

Generation and characterization of human δ-globin-specific monoclonal antibodies

The human delta-globin chain, unique to the hemoglobin A2 (HbA2) heterotetramer, is important for the evaluation of hemoglobinopathy. However, there are no well-defined antibodies specific for the delta-globin chain, a fact that is attributed a striking similarity (93%) in amino acid sequence between delta-globin and beta-globin of the hemoglobin A (HbA). In this study, two monoclonal antibodies (mAbs) against the delta-globin chain were generated and designated as 2H4 and 1H11. These antibodies were specific to HbA2 and do not cross-react with HbA and HbF (fetal hemoglobin). Moreover, the expression of HbA2 in fetal liver and mature erythrocytes was determined using these two mAbs. In addition to being useful tools for research or diagnosis, these antibodies could be valuable for development of rapid and effective antibody-based immunoassays of HbA2 expression in erythroid cells and non-erythroid tissue.

Pretreatment with Mycobacterium avium-derived lipids attenuates the response of murine macrophages to components of Mycobacterium tuberculosis

The high prevalence of Mycobacterium tuberculosis (MTB) despite widely available Bacille Calmette-Guerin (BCG) vaccination may be associated with Mycobacterium avium (M. avium), which may influence the host response to MTB. In this study, we demonstrate that pretreatment of murine macrophages with low-dose Mycobacterium avium-derived lipids (MALs), but not Escherichia coli-derived lipids (ELs), attenuates the clearance of intracellular Mycobacterium bovis BCG (M. bovis BCG) and the production of TNF-α, IL-6, IL-12 and nitric oxide (NO) following stimulation with purified protein derivatives (PPD) or heat-inactivated M. bovis BCG in vitro. Furthermore, a significant decrease in NF-κB activity was observed in MALs-pretreated RAW264.7 cells that were co-transfected with pSV-β-galactosidase and pGL4.32[luc2P/NF-κB-RE/Hygro] prior to stimulation with PPD or heat-inactivated M. bovis BCG. In contrast, IRAK-M, an inhibitor of NF-κB activation, was increased in these cells. This observed hyporesponsiveness is not related to the expression of Toll-like receptor 2 (TLR2). In conclusion, pretreatment with low-dose MALs can induce hyporesponsiveness to MTB components in murine macrophages.