[show abstract][hide abstract] ABSTRACT: The recent approval of a prostate cancer vaccine has renewed hope for anticancer immunotherapies. However, the immunosuppressive
tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to
modulate the tumor microenvironment and improve immunotherapy, but they often are used at high doses in the clinic to prune
tumor vessels and paradoxically may compromise various therapies. Here, we demonstrate that targeting tumor vasculature with
lower vascular-normalizing doses, but not high antivascular/antiangiogenic doses, of an anti-VEGF receptor 2 (VEGFR2) antibody
results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high
doses in polarizing tumor-associated macrophages from an immune inhibitory M2-like phenotype toward an immune stimulatory
M1-like phenotype and in facilitating CD4+ and CD8+ T-cell tumor infiltration. Based on this mechanism, scheduling lower-dose anti-VEGFR2 therapy with T-cell activation induced
by a whole cancer cell vaccine therapy enhanced anticancer efficacy in a CD8+ T-cell–dependent manner in both immune-tolerant and immunogenic murine breast cancer models. These findings indicate that
vascular-normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression
toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy
have not improved overall survival of breast cancer patients, our study suggests a strategy to use antiangiogenic agents in
breast cancer more effectively with active immunotherapy and potentially other anticancer therapies.
Proceedings of the National Academy of Sciences 10/2012; 109(43):17561-17566. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Emerging and re-emerging infectious diseases represent a major challenge to vaccine development since it involves two seemingly contradictory requirements. Rapid and flexible vaccine generation while using technologies and processes that can facilitate accelerated regulatory review. Development in the "-omics" in combination with advances in vaccinology offer novel opportunities to meet these requirements. Here we describe how a consortium of five different organizations from academia and industry is addressing these challenges. This novel approach has the potential to become the new standard in vaccine development allowing timely deployment to avert potential pandemics.
Human vaccines & immunotherapeutics. 07/2012; 8(7):1010-2.
[show abstract][hide abstract] ABSTRACT: The chemokine CXCL12 and its receptor CXCR4 are expressed widely in human cancers, including ovarian cancer, in which they are associated with disease progression at the levels of tumor cell proliferation, invasion, and angiogenesis. Here, we used an immunocompetent mouse model of intraperitoneal papillary epithelial ovarian cancer to show that modulation of the CXCL12/CXCR4 axis in ovarian cancer has multimodal effects on tumor pathogenesis associated with induction of antitumor immunity. siRNA-mediated knockdown of CXCL12 in BR5-1 cells that constitutively express CXCL12 and CXCR4 reduced cell proliferation in vitro, and tumor growth in vivo. Similarly, treatment of BR5-1-derived tumors with AMD3100, a selective CXCR4 antagonist, resulted in increased tumor apoptosis and necrosis, reduction in intraperitoneal dissemination, and selective reduction of intratumoral FoxP3(+) regulatory T cells (Treg). Compared with controls, CXCR4 blockade greatly increased T-cell-mediated antitumor immune responses, conferring a significant survival advantage to AMD3100-treated mice. In addition, the selective effect of CXCR4 antagonism on intratumoral Tregs was associated with both higher CXCR4 expression and increased chemotactic responses to CXCL12, a finding that was also confirmed in a melanoma model. Together, our findings reinforce the concept of a critical role for the CXCL12/CXCR4 axis in ovarian cancer pathogenesis, and they offer a definitive preclinical validation of CXCR4 as a therapeutic target in this disease.
Cancer Research 08/2011; 71(16):5522-34. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: HIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated in immune dysregulatory functions that may limit anti-HIV antigen-specific T cell responses. We hypothesized that in the context of early SHIV infection, immune dysregulation of antigen-specific T-effector cell and regulatory functions would be detectable and that these would be associated or correlated with measurable concentrations of HIV-1gp120 in lymphoid tissues.
Rhesus macaques were intravaginally inoculated with a Clade C CCR5-tropic simian-human immunodeficiency virus, SHIV-1157ipd3N4. HIV-1gp120 levels, antigen-specificity, levels of apoptosis/anergy and frequency and function of Tregs were examined in lymph node and blood derived T cells at 5 and 12 weeks post inoculation.
We observed reduced responses to Gag in CD4 and gp120 in CD8 lymph node-derived T cells compared to the peripheral blood at 5 weeks post-inoculation. Reduced antigen-specific responses were associated with higher levels of PD-1 on lymph node-derived CD4 T cells as compared to peripheral blood and uninfected lymph node-derived CD4 T cells. Lymph nodes contained increased numbers of Tregs as compared to peripheral blood, which positively correlated with gp120 levels; T regulatory cell depletion restored CD8 T cell responses to Gag but not to gp120. HIV gp120 was also able to induce T regulatory cell chemotaxis in a dose-dependent, CCR5-mediated manner. These studies contribute to our broader understanding of the ways in which HIV-1 dysregulates T cell function and localization during early infection.
PLoS ONE 01/2011; 6(4):e18465. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Worldwide, approximately 90% of all human immunodeficiency virus (HIV) transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, then vaginal, and finally oral exposures.
Mucosal lacerations may affect the rank order of susceptibility to HIV but cannot be assessed in humans. We measured relative virus transmissibility across intact mucosae in macaques using a single stock of SHIV-1157ipd3N4, a simian-human immunodeficiency virus encoding a primary R5 HIV clade C env (SHIV-C).
The penetrability of rhesus macaque mucosae differed significantly, with rectal challenge requiring the least virus, followed by vaginal and then oral routes (P = .031, oral vs vaginal; P < .001 rectal vs vaginal). These findings imply that intrinsic mucosal properties are responsible for the differential mucosal permeability. The latter paralleled the rank order reported for humans, with relative risk estimates within the range of epidemiological human studies. To test whether inflammation facilitates virus transmission--as predicted from human studies--we established a macaque model of localized buccal inflammation. Systemic infection occurred across inflamed but not normal buccal mucosa.
Our primate data recapitulate virus transmission risks observed in humans, thus establishing R5 SHIV-1157ipd3N4 in macaques as a robust model system to study cofactors involved in human mucosal HIV transmission and its prevention.
The Journal of Infectious Diseases 03/2010; 201(8):1155-63. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models.
We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone  encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM.
SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.
PLoS ONE 01/2010; 5(7):e11689. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chemoattraction and chemorepulsion are complex directional responses of a cell to external chemotactic stimuli. The decision of a cell to move towards or away from a chemokinetic source includes detection and quantitation of the gradient of the chemotactic agent, biochemical transmission of the stimulus, and translation into a directional migration. This chapter describes a number of in vitro and in vivo assays that can be used to generate and measure both chemoattraction and chemorepulsion of leucocytes. These tools may eventually allow the further characterisation of the mechanism of this complex and physiologically and pathologically important phenomenon.
Methods in molecular biology (Clifton, N.J.) 01/2010; 616:115-24.
[show abstract][hide abstract] ABSTRACT: The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies, yet the molecular effectors of this process are relatively uncharacterized. For extravasation, studies based predominantly on static transwell assays support a general model, whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However, this model of chemotaxis cannot readily reconcile how chemokines influence intravasation, as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus, to analyze how T cells integrate perivascular chemokine signals under physiologic flow, we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment, transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However, when exposedto abluminal CXCL12, transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent, augmented by apical flow, blocked by antibodies to integrins, and reduced by AMD3100 in a dose-dependent manner. Moreover, CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream.
Journal of leukocyte biology 10/2009; 86(6):1285-94. · 4.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human cervical cancer is an immunogenic tumor with a defined pattern of histopathological and clinical progression. Tumor-infiltrating T cells contribute to immune control of this tumor; however, cervical cancer dysregulates this immune response both through its association with human papillomavirus (HPV) infection and by producing cytokines and chemokines. Animal tumor models have revealed associations between overproduction of the chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) and dysregulation of tumor-specific immunity. We therefore proposed that CXCL12 expression by cervical precancerous and cancerous lesions correlates with histopathological progression, loss of immune control of the tumor, and HPV infection. We found a significant association between cancer stage and CXCL12 expression for squamous and glandular lesions as well as with the HPV16+ (high-risk) status of the neoplastic lesions. Cancer progression was correlated with increasing levels of FoxP3 T-cell infiltration in the tumor. FoxP3 and CXCL12 expression significantly correlated for squamous and glandular neoplastic lesions. These observations were supported by enzyme-linked immunosorbent assay and Western blotting. In addition, we demonstrated CXCL12 expression by dyskaryotic cells in ThinPrep cervical smears. This study robustly links increased CXCL12 expression and FoxP3(+)-cell infiltration to HPV infection and progression of cervical cancer. It supports the detection of CXCL12 in cervical smears and biopsies as an additional biomarker for this disease.
American Journal Of Pathology 10/2009; 175(4):1525-35. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to increase the immune breadth of human immunodeficiency virus (HIV) vaccines, strategies such as immunization with several HIV antigens or centralized immunogens have been examined. HIV-1 gp120 protein is a major immunogen of HIV and has been routinely considered for inclusion in both present and future AIDS vaccines. However, recent studies proposed that gp120 interferes with the generation of immune response to codelivered antigens. Here, we investigate whether coimmunization with plasmid-encoded gp120 alters the immune response to other coadministered plasmid encoded antigens such as luciferase or ovalbumin in a mouse model. We found that the presence of gp120 leads to a significant reduction in the expression level of the codelivered antigen in vivo. Antigen presentation by antigen-presenting cells was also reduced and resulted in the induction of weak antigen-specific cellular and humoral immune responses. Importantly, gp120-mediated immune interference was observed after administration of the plasmids at the same or at distinct locations. To characterize the region in gp120 mediating these effects, we used plasmid constructs encoding gp120 that lacks the V1V2 loops (DeltaV1V2) or the V3 loop (DeltaV3). After immunization, the DeltaV1V2, but not the DeltaV3 construct, was able to reduce antigen expression, antigen presentation, and subsequently the immunogenicity of the codelivered antigen. The V3 loop dependence of this phenomenon seems to be limited to V3 loops known to interact with the CXCR4 molecule but not with CCR5. Our study presents a novel mechanism by which HIV-1 gp120 interferes with the immune response against coadministered antigen in a polyvalent vaccine preparation.
Journal of Virology 09/2009; 83(21):10941-50. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The envelope protein of human immunodeficiency virus type 1 (HIV-1)--glycoprotein 120 (gp120)--has been demonstrated to dysregulate T cell function in vitro. We obtained autopsy tissues from individuals with chronic HIV-1 infection to determine whether there was enough gp120 in lymphoid tissues and/or blood to elicit these effects. We found that gp120 was present in high concentrations (>300 pg/mL) in the spleen and lymph nodes of some of these individuals. In contrast, very low amounts of gp120 and p24 were detected in all serum samples tested. These findings underpin the clinical relevance of nonentry functions of gp120 and the chronic nature of human immunodeficiency virus (HIV)-induced immune dysregulation.
The Journal of Infectious Diseases 08/2009; 200(7):1050-3. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gp120 is a critical component of the envelope of HIV-1. Its role in viral entry is well described. In view of its position on the viral envelope, gp120 is a part of the retrovirus that immune cells encounter first and has the potential to influence antiretroviral immune responses. We propose that high levels of gp120 are present in tissues and may contribute to the failure of the immune system to fully control and ultimately clear the virus. Herein, we show for the first time that lymphoid tissues from acutely HIV-1/SIV (SHIV)-KB9-infected macaques contain deposits of gp120 at concentrations that are high enough to induce suppressive effects on T cells, thus negatively regulating the antiviral CTL response and contributing to virus survival and persistence. We also demonstrate that SHIV-KB9 gp120 influences functional T cell responses during SHIV infection in a manner that suppresses degranulation and cytokine secretion by CTLs. Finally, we show that regulatory T cells accumulate in lymphoid tissues during acute infection and that they respond to gp120 by producing TGFbeta, a known suppressant of cytotoxic T cell activity. These findings have significant implications for our understanding of the contribution of non-entry-related functions of HIV-1 gp120 to the pathogenesis of HIV/AIDS.
The Journal of Immunology 11/2008; 181(8):5510-21. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The directional migration of cells within multicellular organisms is governed by gradients of both chemical attractants and repellents in diverse processes, including leukocyte trafficking and neuronal pathfinding in vivo. These complex extracellular environments direct the orchestrated bidirectional trafficking of leukocytes between the vasculature and tissues. Substantial progress has been made in dissecting the molecular mechanisms involved in orchestrating the directed movement of leukocytes into host tissues; however, less is known about the reverse migration of leukocytes from the tissues to the vasculature. In this article, we discuss the functional interplay between chemoattraction and chemorepulsion in the bidirectional movement of cells in complex in vivo environments, and we describe how these mechanisms influence both normal physiology and human disease.
Trends in cell biology 07/2008; 18(6):298-306. · 12.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: The gp160 complex of the envelope of the HIV virus and its component gp120 are essential for viral entry into the host cell. Gp120 binding to its receptor CD4 and co-receptor, CXCR4 or CCR5 is required for fusion of viral and cellular membranes. The presence of gp120 facilitates immune escape of the virus through its profound effect on the immune cells. It is a polyclonal activator of B cells, causing them to differentiate into immunoglobulin producing cells while activating the complement cascade. This results in the formation of immune complexes that are unable to kill the virus but instead shield it from the attack of other immune cells. Such HIV-1 virus that is trapped within immune complexes and is bound to the B cells via CD21 is more infectious than the free virion. In addition, HIV virions are trapped on the membrane of follicular dendritic cells (FDC) processes in immune complexes or through complement receptors. Thus, FDC can serve as a 'Trojan horse' and transmit the trapped virus to CD4+ T lymphocytes as they migrate through the germinal centre to the follicular mantle and paracortical areas. It was demonstrated that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein and its expression by target cells reduces CTL efficacy in vitro. Therefore, apart from the essential role in viral attachment and infection of cells, gp120 possesses several properties that affect the behavior of immune cells and skew the immune response to the virus facilitating viral escape.
Current HIV research 02/2007; 5(1):47-54. · 1.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: The thymic microenvironment provides essential support for the generation of a functional and diverse population of human T cells. In particular, the three-dimensional (3D) thymic architecture contributes to critical cell-cell interactions. We report that thymic stroma, arrayed on a synthetic 3D matrix, supports the development of functional human T cells from hematopoietic precursor cells. Newly generated T cells contain T-cell receptor excision circles and are both fully mature and functional. The coculture of T-cell progenitors with thymic stroma can thus be used to generate de novo functional and diverse T-cell populations. This novel tissue engineered thymic system has biological applications for the study of T-lymphopoiesis and self-tolerance as well as potential therapeutic applications including the immune reconstitution of immunocompromised patients and the induction of tolerance in individuals receiving tissue or organ transplants.
Methods in molecular biology (Clifton, N.J.) 02/2007; 380:163-70.
[show abstract][hide abstract] ABSTRACT: Alloantigen specific T cells have been shown to be required for allograft rejection. The chemokine, stromal cell derived factor-1 (SDF-1) at high concentration, has been shown to act as a T-cell chemorepellent and abrogate T-cell infiltration into a site of antigen challenge in vivo via a mechanism termed fugetaxis or chemorepulsion. We postulated that this mechanism could be exploited therapeutically and that allogeneic cells engineered to express a chemorepellent protein would not be rejected.
Allogeneic murine insulinoma beta-TC3 cells and primary islets from BALB/C mice were engineered to constitutively secrete differential levels of SDF-1 and transplanted into allogeneic diabetic C57BL/6 mice. Rejection was defined as the permanent return of hyperglycemia and was correlated with the level of T-cell infiltration. The migratory response of T-cells to SDF-1 was also analyzed by transwell migration assay and time-lapse videomicroscopy. The cytotoxicity of cytotoxic T cell (CTLs) against beta-TC3 cells expressing high levels of SDF-1 was measured in standard and modified chromium-release assays in order to determine the effect of CTL migration on killing efficacy.
Control animals rejected allogeneic cells and remained diabetic. In contrast, high level SDF-1 production by transplanted cells resulted in increased survival of the allograft and a significant reduction in blood glucose levels and T-cell infiltration into the transplanted tissue.
This is the first demonstration of a novel approach that exploits T-cell chemorepulsion to induce site specific immune isolation and thereby overcomes allograft rejection without the use of systemic immunosuppression.
[show abstract][hide abstract] ABSTRACT: We report for the first time that primary human neutrophils can undergo persistent, directionally biased movement away from a chemokine in vitro and in vivo, termed chemorepulsion or fugetaxis. Robust neutrophil chemorepulsion in microfluidic gradients of interleukin-8 (IL-8; CXC chemokine ligand 8) was dependent on the absolute concentration of chemokine, CXC chemokine receptor 2 (CXCR2), and was associated with polarization of cytoskeletal elements and signaling molecules involved in chemotaxis and leading edge formation. Like chemoattraction, chemorepulsion was pertussis toxin-sensitive and dependent on phosphoinositide-3 kinase, RhoGTPases, and associated proteins. Perturbation of neutrophil intracytoplasmic cyclic adenosine monophosphate concentrations and the activity of protein kinase C isoforms modulated directional bias and persistence of motility and could convert a chemorepellent to a chemoattractant response. Neutrophil chemorepulsion to an IL-8 ortholog was also demonstrated and quantified in a rat model of inflammation. The finding that neutrophils undergo chemorepulsion in response to continuous chemokine gradients expands the paradigm by which neutrophil migration is understood and may reveal a novel approach to our understanding of the homeostatic regulation of inflammation.
Journal of Leukocyte Biology 04/2006; 79(3):539-54. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.
The Journal of Immunology 04/2006; 176(5):2902-14. · 5.52 Impact Factor