Robert Liwski

Cutting Edge: Dendritic Cell Actin Cytoskeletal Polarization during Immunological Synapse Formation Is Highly Antigen-Dependent

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4+ T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4+ T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.

Oral Exposure to Alloantigen Generates Intragraft CD8+ Regulatory Cells

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4+ T cells in fed animals compared with controls, but there was no change in CD8+ T cell numbers. The CD8+ GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients exhibited higher allo-CTL activity than GIC from control unfed recipients. In addition, the CD8+ GIC exhibited high levels of IL-4 mRNA, suggesting Tc2-type regulatory cells. Prolonged graft survival in the face of active CTL and Tc2 cells suggests the presence of a CD8+ regulatory cell population in the allograft. To confirm this, cell transfer experiments were performed. Prolongation of graft survival was transferred from rats orally exposed to alloantigen to naive animals by transfer of CD8+ GIC. This is the first report that oral exposure to alloantigen prolongs kidney allograft survival by the generation of intragraft CD8+ regulatory cells.

Determinants of C1q binding in the single antigen bead assay.

Background A modified single antigen bead (SAB) assay measuring C1q binding to human leukocyte antigen antibodies has recently been introduced. The aim of this study was to investigate the determinants of C1q binding on SAB. Methods Sera from 73 sensitized patients were analyzed by the generic IgGpan as well as IgG subclass specific SAB assays and correlated with the standard and an anti-human globulin (AHG) enhanced C1q test. Results Among 2,665 SABs with positive IgGpan results (mean fluorescence intensity [MFI]>500), strong complement-binding IgG1 and IgG3 subclasses accounted for a median of 99% (interquartile range, 76%–100%) of the total IgG amount. IgGpan MFI alone showed a very strong association with standard C1q positivity (r2=0.72), which was superior to a model including all IgG subclass MFI (r2=0.62). Combining all IgG subclass MFI and IgGpan MFI only marginally improved the prediction of standard C1q positivity compared with IgGpan MFI alone (&Dgr;r2=0.02). IgGpan MFI greater than 14,154 predicted standard C1q positivity, with 92% sensitivity and 96% specificity. Notably, 1,840 (93%) of the 1,974 C1q-negative SABs contained human leukocyte antigen antibodies with strong complement-binding IgG1 and IgG3 subclasses. Anti–human globulin significantly enhanced the signal in the C1q assay, but the association of AHG C1q positivity with IgGpan MFI was less strong (r2=0.51). Conclusion C1q binding on SAB is strongly associated with IgGpan MFI. IgG subclass information only marginally improves prediction of C1q binding likely because complement-binding IgG1 and IgG3 subclasses dominate regarding frequency and relative amounts. A negative C1q assay result does not indicate the absence of strong complement-binding IgG subclasses.

Role of activated protein C and its receptor in inhibition of tumor metastasis.

Engagement of endothelial protein C receptor (EPCR) by activated protein C (aPC) decreases expression of endothelial adhesion molecules implicated in tumor-endothelium interactions. We examined the role of the aPC/EPCR pathway on tumor migration and metastasis. In vitro, B16-F10 melanoma cells showed decreased adhesion to and transmigration through endothelium treated with recombinant human aPC (rhaPC). In murine B16-F10 metastasis models, transgenic EPCR overexpressing (Tie2-EPCR) mice exhibited marked reductions in liver (50%) and lung (92%) metastases compared with wild-type (WT) animals. Intravital imaging showed reduced B16-F10 entrapment within livers of Tie2-EPCR compared with WT mice. A similar reduction was observed in WT mice treated with rhaPC. Strikingly, rhaPC treatment resulted in a 44% reduction in lung metastases. This was associated with decreased lung P-selectin and TNF-alpha mRNA levels. These findings support an important role for the aPC/EPCR pathway in reducing metastasis via inhibition of tumor cell adhesion and transmigration.

Optimized cell transplantation using adult rag2 mutant zebrafish

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2E450fs mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16‐F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline‐treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16‐F10 cells exhibited decreased interaction with platelets from ticagrelor‐treated mice compared to saline‐treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16‐F10 cells co‐incubated with platelets from ticagrelor‐treated mice exhibited reduced adhesion to endothelial monolayers compared to those co‐incubated with platelets from saline‐treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12‐mediated platelet activation in promoting metastases, and provide proof‐of‐concept for the clinical use of ticagrelor in the prevention of tumor metastasis.

Piperine, a dietary phytochemical, inhibits angiogenesis

Angiogenesis plays an important role in tumor progression. Piperine, a major alkaloid constituent of black pepper, has diverse physiological actions including killing of cancer cells; however, the effect of piperine on angiogenesis is not known. Here we show that piperine inhibited the proliferation and G(1)/S transition of human umbilical vein endothelial cells (HUVECs) without causing cell death. Piperine also inhibited HUVEC migration and tubule formation in vitro, as well as collagen-induced angiogenic activity by rat aorta explants and breast cancer cell-induced angiogenesis in chick embryos. Although piperine binds to and activates the cation channel transient receptor potential vanilloid 1 (TRPV1), its effects on endothelial cells did not involve TRPV1 since the antiproliferative effect of piperine was not affected by TRPV1-selective antagonists, nor did HUVECs express detectable TRPV1 mRNA. Importantly, piperine inhibited phosphorylation of Ser 473 and Thr 308 residues of Akt (protein kinase B), which is a key regulator of endothelial cell function and angiogenesis. Consistent with Akt inhibition as the basis of piperines action on HUVECs, inhibition of the phosphoinositide-3 kinase/Akt signaling pathway with LY-294002 also inhibited HUVEC proliferation and collagen-induced angiogenesis. Taken together, these data support the further investigation of piperine as an angiogenesis inhibitor for use in cancer treatment.

Immune Modulation by Chemotherapy or Immunotherapy to Enhance Cancer Vaccines

Chemotherapy has been a mainstay in cancer treatment for many years. Despite some success, the cure rate with chemotherapy remains unsatisfactory in some types of cancers, and severe side effects from these treatments are a concern. Recently, understanding of the dynamic interplay between the tumor and immune system has led to the development of novel immunotherapies, including cancer vaccines. Cancer vaccines have many advantageous features, but their use has been hampered by poor immunogenicity. Many developments have increased their potency in pre-clinical models, but cancer vaccines continue to have a poor clinical track record. In part, this could be due to an inability to effectively overcome tumor-induced immune suppression. It had been generally assumed that immune-stimulatory cancer vaccines could not be used in combination with immunosuppressive chemotherapies, but recent evidence has challenged this dogma. Chemotherapies could be used to condition the immune system and tumor to create an environment where cancer vaccines have a better chance of success. Other types of immunotherapies could also be used to modulate the immune system. This review will discuss how immune modulation by chemotherapy or immunotherapy could be used to bolster the effects of cancer vaccines and discuss the advantages and disadvantages of these treatments.

Technical aspects of Hla antibody testing

Purpose of review Since the landmark studies of Patel and Terasaki, pretransplant identification of donor-directed HLA alloantibodies (DSAs) has been a critical prelude to renal allograft transplantation. Pretransplant, DSAs may be an acceptable risk or an unconditional contraindication to transplantation depending on the particular donor : recipient combination. Posttransplant, DSAs are associated with episodes of acute rejection, chronic rejection, and graft loss. Thus, monitoring for such antibodies is an important aspect of patient care. Recent findings The development of solid-phase antibody detection assays significantly enhanced our ability to identify HLA antibodies, taking virtual crossmatching from concept to reality. At the root of these detection assays are two questions that have been asked for almost 50 years: are donor-directed HLA antibodies present and, if so, are they clinically relevant? While the technology related to solid-phase antibody detection has seemingly allowed the first question to be answered with exquisite sensitivity and specificity, can the same be said for question 2? Summary Solid-phase antibody detection assays have clear benefits over historical approaches to antibody identification, but are not flawless. In fact, the limitations of these assays are frequently ignored. Herein, the strengths and weaknesses of solid-phase antibody detection are highlighted.

Prolongation of allograft survival by Nippostrongylus brasiliensis is associated with decreased allospecific cytotoxic T lymphocyte activity and development of T cytotoxic cell type 2 cells.

BACKGROUND We have demonstrated that infection with Nippostrongylus brasiliensis (Nb), which induces strong type 2 responses, prolongs kidney allograft survival in rats. Here, we confirm that this effect is not species-specific and address immune modulation in allospecific T-cell responses mediated by nematode infection. METHODS C57BL/6 mice were injected with Nb or phosphate-buffered saline. Four days later, mice were transplanted with BALB/c hearts and graft survival was assessed. In other experiments, Nb-infected mice were immunized with BALB/c spleen cells and allospecific T-cell responses were determined in vitro. RESULTS In this study, we show that Nb prolongs cardiac allograft survival in mice. Further, spleen T cells from Nb-infected, allo-immunized mice exhibit reduced allospecific cytotoxic T-lymphocyte activity. In contrast, allospecific proliferation of T cells in the mixed lymphocyte reaction was not reduced by Nb, ruling out immunosuppression as the mechanism of Nb-induced allograft survival. Nb infection induced IL-4 and IL-6 and inhibited IFN-gamma production by T cells in response to allo-antigen. Furthermore, anti-IL-4 treatment reduced allospecific T-cell proliferation from Nb-infected but not control mice, indicating that type 2 allospecific T cells develop in the presence of Nb. We also double-stained T cells for CD8 and IL-4 and showed that Nb induces an 8-fold increase in Tc2 cell numbers. CONCLUSIONS These results are consistent with a hypothesis that Nb mediates prolongation of allograft survival through induction of type 2 immunity, including the development of regulatory Tc2 cells, and subsequent inhibition of allospecific cytotoxic T-lymphocyte activity.