Avi-Hai Hovav

Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

Thy1+ Nk Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1+ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.

The Mycobacterium tuberculosis Recombinant 27-Kilodalton Lipoprotein Induces a Strong Th1-Type Immune Response Deleterious to Protection

ABSTRACT Th1 immune response is essential in the protection against mycobacterial intracellular pathogens. Lipoproteins trigger both humoral and cellular immune responses and may be candidate protective antigens. We studied in BALB/c mice the immunogenicity and the protection offered by the recombinant 27-kDa Mycobacterium tuberculosis lipoprotein and the corresponding DNA vaccine. Immunization with the 27-kDa antigen resulted in high titers of immunoglobulin G1 (IgG1) and IgG2a with a typical Th1 profile and a strong delayed hypersensitivity response. A strong proliferation response was observed in splenocytes, and significant nitric oxide production and gamma interferon secretion but not interleukin 10 secretion were measured. Based on these criteria, the 27-kDa antigen induced a typical Th1-type immune response thought to be necessary for protection. Surprisingly, in 27-kDa-vaccinated mice (protein or DNA vaccines) challenged by M. tuberculosis H37Rv or BCG strains, there was a significant increase in the numbers of CFU in the spleen compared to that for control groups. Furthermore, the protection provided by BCG or other mycobacterial antigens was completely abolished once the 27-kDa antigen was added to the vaccine preparations. This study indicates that the 27-kDa antigen has an adverse effect on the protection afforded by recognized vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response.

Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages

Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human alpha1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.

Generation of CD8+ T-Cell Responses by a Recombinant Nonpathogenic Mycobacterium smegmatis Vaccine Vector Expressing Human Immunodeficiency Virus Type 1 Env

ABSTRACT Because the vaccine vectors currently being evaluated in human populations all have significant limitations in their immunogenicity, novel vaccine strategies are needed for the elicitation of cell-mediated immunity. The nonpathogenic, rapidly growing mycobacterium Mycobacterium smegmatis was engineered as a vector expressing full-length human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope protein. Immunization of mice with recombinant M. smegmatis led to the expansion of major histocompatibility complex class I-restricted HIV-1 epitope-specific CD8+ T cells that were cytolytic and secreted gamma interferon. Effector and memory T lymphocytes were elicited, and repeated immunization generated a stable central memory pool of virus-specific cells. Importantly, preexisting immunity to Mycobacterium bovis BCG had only a marginal effect on the immunogenicity of recombinant M. smegmatis. This mycobacterium may therefore be a useful vaccine vector.

Duration of Antigen Expression In Vivo following DNA Immunization Modifies the Magnitude, Contraction, and Secondary Responses of CD8+ T Lymphocytes

The duration of Ag expression in vivo has been reported to have a minimal impact on both the magnitude and kinetics of contraction of a pathogen-induced CD8+ T cell response. In this study, we controlled the duration of Ag expression by excising the ear pinnae following intradermal ear pinnae DNA immunization. This resulted in decreased magnitude, accelerated contraction and differentiation, and surprisingly greater secondary CD8+ T cell responses. Furthermore, we found delayed and prolonged Ag presentation in the immunized mice; however, this presentation was considerably decreased when the depot Ag was eliminated. These findings suggest that the magnitude and the contraction phase of the CD8+ T cell response following intradermal DNA immunization is regulated by the duration rather than the initial exposure to Ag.

The immunogenicity of Mycobacterium paratuberculosis 85B antigen

Abstract.Mycobacterium paratuberculosis (MPT) is the etiological agent of paratuberculosis. The disease is prevalent throughout the world, and exacts a heavy financial toll. At present, the only means of controlling this disease are culling or vaccination. The existing vaccines are not very efficient and produce a long-lasting local reaction at the point of injection and induce antibodies/delayed-type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are necessary to improve the control of this disease. We have isolated, overexpressed and purified the 85B antigen of MPT, and characterized the immune response induced by this antigen in mice. Our results showed that the recombinant MPT 85B (rMPT 85B) antigen induced a high production of interferon (IFN)γ, interleukin (IL)-6, IL-10 and nitric oxide (NO). Spleen cells from mice immunized with rMPT 85B in Ribi adjuvant produced a higher level of IL-10 and NO than spleen cells of mice immunized with rMPT 85B only. In contrast, the addition of Ribi to the immunization protocol resulted in a lower amount of IFNγ released by spleen cells. The levels of spleen cells proliferation in mice vaccinated with the rMPT 85B protein alone or with rMPT 85B with Ribi adjuvant were, respectively, four times or five times greater than in the control mice. The Ribi adjuvant induced significantly higher anti-85B antibody production of all classes tested and increased the IgG1/IgG2a ratio. DTH responses in mice footpads were observed only in mice immunized with rMPT 85B emulsified in Ribi. rMPT 85B induced both a Th1 and Th2 type of immune response with the later slightly more pronounced when the vaccination protocol comprised Ribi as an adjuvant. The rMPT 85B antigen elicited a strong immune response and can be considered as a potential candidate for a future acellular vaccine.

Langerhans cells down-regulate inflammation-driven alveolar bone loss

Excessive bone resorption is frequently associated with chronic infections and inflammatory diseases. Whereas T cells were demonstrated to facilitate osteoclastogenesis in such diseases, the role of dendritic cells, the most potent activators of naive T cells, remains unclear. Using a model involving inflammation-driven alveolar bone loss attributable to infection, we showed that in vivo ablation of Langerhans cells (LCs) resulted in enhanced bone loss. An increased infiltration of B and T lymphocytes into the tissue surrounding the bone was observed in LC-ablated mice, including receptor activator of NF-κB ligand (RANKL)-expressing CD4+ T cells with known capabilities of altering bone homeostasis. In addition, the absence of LCs significantly reduced the numbers of CD4+Foxp3+ T-regulatory cells in the tissue. Further investigation revealed that LCs were not directly involved in presenting antigens to T cells. Nevertheless, despite their low numbers in the tissue, the absence of LCs resulted in an elevated activation of CD4+ but not CD8+ T cells. This activation involved elevated production of IFN-γ but not IL-17 or IL-10 cytokines. Our data, thus, reveal a protective immunoregulatory role for LCs in inflammation-induced alveolar bone resorption, by inhibiting IFN-γ secretion and excessive activation of RANKL+CD4+ T cells with a capability of promoting osteoclastogenesis.

The Role of Skin-Derived Dendritic Cells in CD8+ T Cell Priming Following Immunization with Lentivectors

Although skin dendritic cells (DCs) have been shown to directly present Ag to CD8+ T cells after intradermal immunization with lentivectors, the contribution of the different skin DC subsets to this process remains unclear. Using langerin-diphtheria toxin receptor transgenic mice we demonstrated that ablation of langerhans cells and langerin-expressing positive dermal DCs (Ln+dDCs) did not interfere with the generation of CD8+ T cells by lentiviral vectors. Consistent with these findings, the absence of langerhans cells and Ln+dDCs did not hamper the presentation level of lentiviral-derived Ag by skin DCs in vitro. We further demonstrated that only dDCs and Ln+dDCs were capable of presenting Ag, however, the number of dDCs migrating to the draining lymph nodes was 6-fold higher than that of Ln+dDCs. To study how the duration of DC migration influences CD8+ T cell responses, we analyzed the kinetics of Ag expression at the injection site and manipulated DC migration by excising the injected skin at various times after immunization. A low level of Ag expression was seen 1 wk after the immunization; peaked during week 2, and was considerably cleared by week 3 via a perforin-dependent fas-independent mechanism. Removing the injection site 3 or 5 d, but not 10 d, after the immunization, resulted in a reduced CD8+ T cell response. These findings suggest that dDCs are the main APCs active after intradermal lentiviral-mediated immunization, and migration of dDCs in the initial 10-d period postimmunization is required for optimal CD8+ T cell induction.

The Impact of a Boosting Immunogen on the Differentiation of Secondary Memory CD8+ T Cells

ABSTRACT While recent studies have demonstrated that secondary CD8+ T cells develop into effector-memory cells, the impact of particular vaccine regimens on the elicitation of these cells remains poorly defined. In the present study we evaluated the effect of three different immunogens—recombinant vaccinia, recombinant adenovirus, and plasmid DNA—on the generation of memory cellular immune responses. We found that vectors that induce the rapid movement of CD8+ T cells into the memory compartment during a primary immune response also drive a rapid differentiation of these cells into effector-memory CD8+ T cells following a secondary immunization. In contrast, the functional profiles of both CD8+ and CD4+ T cells, assessed by measuring antigen-stimulated gamma interferon and interleukin-2 production, were not predominantly shaped by the boosting immunogen. We also demonstrated that the in vivo expression of antigen by recombinant vectors was brief following boosting immunization, suggesting that antigen persistence has a minimal impact on the differentiation of secondary CD8+ T cells. When used in heterologous or in homologous prime-boost combinations, these three vectors generated antigen-specific CD8+ T cells with different phenotypic profiles. Expression of the memory-associated molecule CD27 on effector CD8+ T cells decreased following heterologous but not homologous boosting, resulting in a phenotypic profile similar to that seen on primary CD8+ T cells. These data therefore suggest that the phenotype of secondary CD8+ T cells is determined predominantly by the boosting immunogen whereas the cytokine profile of these cells is shaped by both the priming and boosting immunogens.