Bennett D. Elzey

Platelet-Mediated Modulation of Adaptive Immunity: A Communication Link between Innate and Adaptive Immune Compartments

Platelets are highly reactive components of the circulatory system with well-documented hemostatic function. Recent studies extend platelet function to modulation of local inflammatory events through the release of chemokines, cytokines, and a number of immunomodulatory ligands, including CD154. We hypothesized that platelet-derived CD154 modulates adaptive immunity. The data reported herein demonstrate that platelets, via CD154, induce dendritic cell maturation, B cell isotype switching, and augment CD8(+) T cell responses both in vitro and in vivo. Platelet transfusion studies demonstrate that platelet-derived CD154 alone is sufficient to induce isotype switching and augment T lymphocyte function during viral infection, leading to enhanced protection against viral rechallenge. Additionally, depletion of platelets in normal mice results in decreased antigen-specific antibody production.

Uptake of apoptotic antigen-coupled cells by lymphoid dendritic cells and cross-priming of CD8(+) T cells produce active immune unresponsiveness.

The induction of immunologic unresponsiveness by i.v. administration of Ag-coupled lymphoid cells has been studied extensively, but the mechanisms remain unclear. We have further explored this model by examining the role of Fas/Fas ligand (FasL)-mediated apoptosis. Using i.v. injection of trinitrophenyl-coupled splenocytes (TNP-spl) as tolerogen, we found that Fas signaling for apoptosis in the spleen cells delivered by FasL in the recipient is the critical event. The requirement for Fas and FasL was overcome by prior induction of apoptosis in TNP-spl, making the tolerogen 100 times more potent. Prevention of apoptosis by a caspase inhibitor blocks tolerance. Interestingly, while blocking CD40/CD40 ligand interaction does not prevent tolerance induction, an agonist anti-CD40 Ab turns tolerogenic TNP-spl into an immunizing Ag. Studies further showed that tolerance is induced through cross-presentation of Ag in a class I MHC-dependent manner by CD8+CD11c+ lymphoid-derived dendritic cells to regulatory T cells. The results provide a mechanism for a well-established method of inducing immunologic unresponsiveness.

Platelet-mediated modulation of adaptive immunity: unique delivery of CD154 signal by platelet-derived membrane vesicles

Although mounting evidence indicates that platelets participate in the modulation of both innate and adaptive immunity, the mechanisms by which platelets exert these effects have not been clearly defined. The study reported herein uses a previously documented adoptive transfer model to investigate the ability of platelet-derived membrane vesicles to communicate activation signals to the B-cell compartment. The findings demonstrate for the first time that platelet-derived membrane vesicles are sufficient to deliver CD154 to stimulate antigen-specific IgG production and modulate germinal center formation through cooperation with responses elicited by CD4(+) T cells. The data are consistent with the hypothesis that platelets modulate inflammation and adaptive immunity at sites distant from the location of activation and that platelet-derived membrane vesicles are sufficient to mediate the effect.

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (alvac) cytokine gene delivery induces destruction of established prostate tumors

Prostate‐specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5‐PSA) that is safe and effectively activates a PSA‐specific T‐cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5‐PSA induced PSA‐specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock‐transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding β‐galactosidase (Ad5‐lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8+ T lymphocytes. Although Ad5‐PSA immunization prior to RM11psa challenge was protective, Ad5‐PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm3 were efficiently eliminated if Ad5‐PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin‐12 (IL‐12), IL‐2, and tumor necrosis factor‐α. In this case, antitumor immunity was still dominated by CD8+ T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5‐PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods.

Cooperation between platelet-derived CD154 and CD4+ T cells for enhanced germinal center formation

It has been demonstrated previously that platelet‐derived CD154 communicates with the adaptive immune compartment, enhancing B and T cell responses in CD154−/− mice. The presence of platelets was also shown to be necessary for optimal production of immunoglobulin G (IgG) in normal C57BL/6 mice. These data led us to hypothesize that platelets perform a sentinel function, quickly relaying activating signals to the adaptive immune compartment. Here, we report that platelet‐derived CD154 increases serum IgG levels and germinal center formation under conditions where antigen‐specific CD4+ T cell numbers are limiting. We propose that in the physiologic setting where antigen‐specific B and T cells are rare, platelets function to enhance signals required for robust adaptive humoral immunity.

Plasmacytoid Dendritic Cell-Derived IFN-α Induces TNF-Related Apoptosis-Inducing Ligand/Apo-2L-Mediated Antitumor Activity by Human Monocytes Following CpG Oligodeoxynucleotide Stimulation

Immunostimulatory oligodeoxynucleotides (ODN) containing the CpG motif are being tested as immune adjuvants in many disease settings. Of the human PBMC examined, plasmacytoid dendritic cells (pDC) are a major source of type I IFN upon stimulation with CpG ODN. IFNs have numerous immunostimulatory effects, including the induction of TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L on monocytes, NK cells, and T cells. Importantly, IFN has also been linked to antitumor responses. Thus, we tested whether CpG ODN stimulation of PBMC led to TRAIL/Apo-2L-induced tumor cell death. When PBMC were stimulated with CpG ODN, TRAIL/Apo-2L-dependent tumor cell death was observed. Further examination of CpG ODN-stimulated PBMC revealed that TRAIL/Apo-2L expression was limited to CD14+ cells, which, when depleted, led to a loss of the TRAIL/Apo-2L-mediated tumor cell killing. Moreover, pDC depletion also abolished the TRAIL/Apo-2L-mediated killing of tumor cell targets. Analysis of the pDC showed IFN-α production after CpG ODN stimulation. Finally, inclusion of neutralizing IFN-α antiserum with the PBMC during CpG ODN stimulation abrogated TRAIL/Apo-2L-mediated tumor cell killing. These results define a mechanism by which CpG ODN induces TRAIL/Apo-2L-dependent killing of tumor cells by CD14+ PBMC, in which CpG ODN-activated pDC produce IFN-α that stimulates CD14+ PBMC to express functional TRAIL/Apo-2L.

Cutting Edge: Restoration of the Ability to Generate CTL in Mice Immune to Adenovirus by Delivery of Virus in a Collagen-Based Matrix

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.

Regulation of Fas Ligand-Induced Apoptosis by TNF

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas+ lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2−/− T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1−/− T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1−/− 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2−/− 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.

The fibronectin attachment protein of bacillus Calmette-Guerin (BCG) mediates antitumor activity

PurposeThe receptor responsible for the attachment of bacillus Calmette-Guerin (BCG) to fibronectin, fibronectin attachment protein (FAP), has been cloned. Studies targeting FAP as an inducer of immunity in mycobacterial infections suggest that FAP is a highly immunogenic protein. In light of these findings and the need to find effective alternatives to BCG treatment for bladder cancer, we tested the ability of FAP to induce antitumor activity.Materials and methodsThe ability of FAP to bind to bladder tumor cells and the bladder wall was established using 125I-FAP. For testing antitumor activity in vivo, mice were catheterized and 5xa0×xa0104 MB-49 bladder tumor cells were implanted orthotopically on day 0. Test groups were treated with PBS only, FAP, or BCG on day 1 and day 8. A subset of mice was preimmunized with FAP prior to treatment.ResultsFAP was observed to bind to bladder tumor cells in a fibronectin-dependent manner. Attachment of FAP within the bladder followed the pattern established for BCG binding. Antitumor studies showed a significant reduction in tumor growth in FAP-treated mice that had been preimmunized with FAP. Tumor growth was not inhibited in naïve mice treated with FAP. Dose-response studies showed that FAP-induced antitumor activity is dose dependent, and experiments comparing BCG with FAP showed equivalent antitumor effects. In vitro experiments showed antigen-specific lymphocyte proliferation and a cytokine profile indicative of Th-1 polarization of the FAP-induced immune response. CD8+ T cells and natural killer cells were found to be required for the FAP-induced antitumor response.ConclusionsFAP is an effective antitumor agent that inhibits tumor growth at a level equivalent to that observed for BCG. This protein may thus provide an alternative to BCG for treatment of superficial bladder cancer.

Nontoxic Formulations of Scintillation Nanocrystals for Use as X-ray Computed Tomography Contrast Agents

X-ray computed tomography (CT) is currently one of the most powerful, noninvasive, clinical in vivo imaging techniques, which has resulted from advances in both X-ray device and contrast enhancement technologies. The present study demonstrates, for the first time, that metal tungstates (such as CaWO4) are promising contrast agents for X-ray, radiation, and CT imaging, because of the high X-ray mass attenuation of tungsten (W). We have developed a method of formulation, in which CaWO4 (CWO) nanoparticles (NPs) are encapsulated within a biocompatible poly(ethylene glycol-b-d,l-lactic acid) (PEG-PLA) block copolymer (BCP) capsule. We show that these PEG-PLA-encapsulated CWO NPs (170 ± 10 nm hydrodynamic diameter) produce a higher CT contrast (by a factor of about 2) than commercial iodine-based radiocontrast agents (e.g., Iohexol) at identical molar concentrations of W or I atoms. PEG-PLA-coated CWO NPs are chemically stable and completely nontoxic. It was confirmed that the maximum tolerated dose (MTD) of this material in mice is significantly higher (250 ± 50 mg per kg body weight following a single intravenous (IV) administration) than, for instance, commercially available dextran-coated iron oxide nanoparticles that are currently used clinically as MRI contrast agents (MTD in mice ≈ 168 mg/kg per dose IV). IV-injected PEG-PLA/CWO NPs caused no histopathologic damage in major excretory organs (heart, liver, lungs, spleen, and kidney). When an IV dose of 100 mg/kg was given to mice, the blood circulation half-life was measured to be about 4 h, and more than 90% of the NPs were cleared from the mice within 24 h via the renal and hepatobiliary systems. When intratumorally administered, PEG-PLA-coated CWO NPs showed complete retention in a tumor-bearing mouse model (measurements were made up to 1 week). These results suggest that PEG-PLA-coated CWO NPs are promising materials for use in CT contrast.