Xavier Alvarez

Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival

Regulatory T (Treg) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Failure of host antitumor immunity may be caused by exaggerated suppression of tumor-associated antigen–reactive lymphocytes mediated by Treg cells; however, definitive evidence that Treg cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4+CD25+FOXP3+ Treg cells in 104 individuals affected with ovarian carcinoma, that human tumor Treg cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor Treg cells are associated with a high death hazard and reduced survival. Human Treg cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages. Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of Treg cells to the tumor. This specific recruitment of Treg cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking Treg cell migration or function may help to defeat human cancer.

Blockade of B7-H1 improves myeloid dendritic cell–mediated antitumor immunity

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-γ. T cells conditioned with the B7-H1–blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic–severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.

B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma

Tumor-associated macrophages are a prominent component of ovarian cancer stroma and contribute to tumor progression. B7-H4 is a recently identified B7 family molecule. We show that primary ovarian tumor cells express intracellular B7-H4, whereas a fraction of tumor macrophages expresses surface B7-H4. B7-H4+ tumor macrophages, but not primary ovarian tumor cells, suppress tumor-associated antigen-specific T cell immunity. Blocking B7-H4-, but not arginase-, inducible nitric oxide synthase or B7-H1 restored the T cell stimulating capacity of the macrophages and contributes to tumor regression in vivo. Interleukin (IL)-6 and IL-10 are found in high concentrations in the tumor microenvironment. These cytokines stimulate macrophage B7-H4 expression. In contrast, granulocyte/macrophage colony-stimulating factor and IL-4, which are limited in the tumor microenvironment, inhibit B7-H4 expression. Ectopic expression of B7-H4 makes normal macrophages suppressive. Thus, B7-H4+ tumor macrophages constitute a novel suppressor cell population in ovarian cancer. B7-H4 expression represents a critical checkpoint in determining host responses to dysfunctional cytokines in ovarian cancer. Blocking B7-H4 or depleting B7-H4+ tumor macrophages may represent novel strategies to enhance T cell tumor immunity in cancer.

Dendritic Cell Subsets Differentially Regulate Angiogenesis in Human Ovarian Cancer

Angiogenesis is essential for both primary and metastatic tumor growth. Tumor blood vessel formation is complex and regulated by many factors. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites of patients with untreated ovarian carcinoma. We observed high numbers of plasmacytoid dendritic cells (PDCs) and significant stromal-derived factor (CXCL-12/SDF)-1 in their malignant ascites, attracting PDCs into the tumor environment. We now show that tumor-associated PDCs induced angiogenesis in vivo through production of tumor necrosis factor α and interleukin 8. By contrast, myeloid dendritic cells (MDCs) were absent from malignant ascites. MDCs derived in vitro suppressed angiogenesis in vivo through production of interleukin 12. Thus, the tumor may attract PDCs to augment angiogenesis while excluding MDCs to prevent angiogenesis inhibition, demonstrating a novel mechanism for modulating tumor neovascularization. Because dendritic cells (DCs) have long been known to affect tumor immunity, our data also implicate DCs in regulation of tumor neoangiogenesis, suggesting a novel role of DCs in tumor pathology.

Increased Monocyte Turnover from Bone Marrow Correlates with Severity of SIV Encephalitis and CD163 Levels in Plasma

Cells of the myeloid lineage are significant targets for human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in monkeys. Monocytes play critical roles in innate and adaptive immunity during inflammation. We hypothesize that specific subsets of monocytes expand with AIDS and drive central nervous system (CNS) disease. Additionally, there may be expansion of cells from the bone marrow through blood with subsequent macrophage accumulation in tissues driving pathogenesis. To identify monocytes that recently emigrated from bone marrow, we used 5-bromo-2′-deoxyuridine (BrdU) labeling in a longitudinal study of SIV-infected CD8+ T lymphocyte depleted macaques. Monocyte expansion and kinetics in blood was assessed and newly migrated monocyte/macrophages were identified within the CNS. Five animals developed rapid AIDS with differing severity of SIVE. The percentages of BrdU+ monocytes in these animals increased dramatically, early after infection, peaking at necropsy where the percentage of BrdU+ monocytes correlated with the severity of SIVE. Early analysis revealed changes in the percentages of BrdU+ monocytes between slow and rapid progressors as early as 8 days and consistently by 27 days post infection. Soluble CD163 (sCD163) in plasma correlated with the percentage of BrdU+ monocytes in blood, demonstrating a relationship between monocyte activation and expansion with disease. BrdU+ monocytes/macrophages were found within perivascular spaces and SIVE lesions. The majority (80–90%) of the BrdU+ cells were Mac387+ that were not productively infected. There was a minor population of CD68+BrdU+ cells (<10%), very few of which were infected (<1% of total BrdU+ cells). Our results suggest that an increased rate of monocyte recruitment from bone marrow into the blood correlates with rapid progression to AIDS, and the magnitude of BrdU+ monocytes correlates with the severity of SIVE.

Genetic Requirements for the Survival of Tubercle Bacilli in Primates

BACKGROUND Tuberculosis (TB) leads to the death of 1.7 million people annually. The failure of the bacille Calmette-Guérin vaccine, synergy between AIDS and TB, and the emergence of drug resistance have worsened this situation. It is imperative to delineate the mechanisms employed by Mycobacterium tuberculosis to successfully infect and persist in mammalian lungs. METHODS Nonhuman primates (NHPs) are arguably the best animal system to model critical aspects of human TB. We studied genes essential for growth and survival of M. tuberculosis in the lungs of NHPs experimentally exposed to aerosols of an M. tuberculosis transposon mutant library. RESULTS Mutants in 108 M. tuberculosis genes (33.13% of all genes tested) were attenuated for in vivo growth. Comparable studies have reported the attenuation of only approximately 6% of mutants in mice. The M. tuberculosis mutants attenuated for in vivo survival in primates were involved in the transport of various biomolecules, including lipid virulence factors; biosynthesis of cell-wall arabinan and peptidoglycan; DNA repair; sterol metabolism; and mammalian cell entry. CONCLUSIONS Our study highlights the various virulence mechanisms employed by M. tuberculosis to overcome the hostile environment encountered during infection of primates. Prophylactic approaches aimed against bacterial factors that respond to such in vivo stressors have the potential to prevent infection at an early stage, thus likely reducing the extent of transmission of M. tuberculosis.

The level of monocyte turnover predicts disease progression in the macaque model of AIDS

It is widely accepted that destruction of CD4(+) T cells and viral load are the primary markers for immunodeficiency in HIV-1-infected humans and in simian immunodeficiency virus (SIV)-infected macaques. However, monocyte/macrophages are also important targets of HIV/SIV infection and a critical link between innate and adaptive immunity. We therefore examined whether changes in cells of the monocyte/macrophage lineage could be linked to the pathogenesis of AIDS in the rhesus macaque model. Here, we show that massive turnover of peripheral monocytes associated with death of tissue macrophages correlates with AIDS progression in macaques. More importantly, the level of monocyte turnover was not linked to the CD4(+) T-cell count and was a better predictive marker for AIDS progression than was viral load or lymphocyte activation. Our results show the importance of monocyte/macrophages in the pathogenesis of AIDS and suggest the dynamic changes of the monocyte/macrophages as a new marker for AIDS progression.

CD163, a Marker of Perivascular Macrophages, Is Up-Regulated by Microglia in Simian Immunodeficiency Virus Encephalitis after Haptoglobin-Hemoglobin Complex Stimulation and Is Suggestive of Breakdown of the Blood-Brain Barrier

Macrophages and microglia are the major cell types infected by human immunodeficiency virus and simian immunodeficiency virus (SIV) in the central nervous system. Microglia are likely infected in vivo, but evidence of widespread productive infection (ie, presence of viral RNA and protein) is lacking. This conclusion is controversial because, unlike lymphocytes, macrophages and microglia cannot be discreetly immunophenotyped. Of particular interest in the search for additional monocyte/macrophage-lineage cell markers is CD163; this receptor for haptoglobin-hemoglobin (Hp-Hb) complex, which forms in plasma following erythrolysis, is expressed exclusively on cells of monocyte/macrophage lineage. We examined CD163 expression in vitro and in vivo by multiple techniques and at varying times after SIV infection in macaques with or without encephalitis. In normal and acutely SIV-infected animals, and in SIV-infected animals without encephalitis, CD163 expression was detected in cells of monocyte/macrophage lineage, including perivascular macrophages, but not in parenchymal microglia. However, in chronically infected animals with encephalitis, CD163 expression was detected in activated microglia surrounding SIV encephalitis lesions in the presence of Hp-Hb complex, suggesting leakage of the blood-brain barrier. CD163 expression was also induced on microglia in vitro after stimulation with Hp-Hb complex. We conclude that CD163 is a selective marker of perivascular macrophages in normal macaques and during the early phases of SIV infection; however, later in infection in animals with encephalitis, CD163 is also expressed by microglia, which are probably activated as a result of vascular compromise.

Monocyte/macrophage traffic in HIV and SIV encephalitis.

This short review focuses on the role of central nervous system (CNS) perivascular macrophages as targets of productive infection of the CNS. Data discussed include the importance of these cells as early targets of infection and their productive infection with AIDS. Many of the immune molecules on perivascular macrophages are also found on subsets of blood monocyte/macrophages, some of which are expanded during human immunodeficiency virus (HIV) infection. These observations paired with the known bone marrow (BM) origin of perivascular macrophages and the BM as a site of HIV infection underscore the importance of the study of monocyte populations in the BM and blood, which are activated and infected as a source of virus that enters the CNS. Data presented and discussed herein suggest a role of HIV‐infected BM‐derived monocytes as “Trojan horse” cells that traffic to the CNS to become perivascular macrophages. The study of such cells including their timing of infection, activation, and traffic and the role of HIV‐specific immune responses controlling their accumulation in the CNS warrant study with regard to CNS neuropathogenesis.

Both Mucosal and Systemic Routes of Immunization with the Live, Attenuated NYVAC/Simian Immunodeficiency Virus SIVgpe Recombinant Vaccine Result in Gag-Specific CD8+ T-Cell Responses in Mucosal Tissues of Macaques

ABSTRACT As most human immunodeficiency virus (HIV) infection occurs via mucosal surfaces, an important goal of vaccination may be the induction of virus-specific immune responses at mucosal sites to contain viral infection early on. Here we designed a study in macaques carrying the major histocompatibility complex class I Mamu-A∗01 molecule to assess the capacity of the highly attenuated poxvirus NYVAC/simian immunodeficiency virus (SIV) SIVgpe vaccine candidate administered by the intranasal, intramuscular, or intrarectal route to induce mucosal immunity. All macaques, including one naive macaque, were exposed to SIVmac251 by the intrarectal route and sacrificed 48 h after infection. The kinetics of immune response at various time points following immunization with NYVAC/SIVgpe and the anamnestic response to SIVmac251 at 48 h after challenge were assessed in blood, in serial rectal and vaginal biopsy samples, and in tissues at euthanasia with an SIVmac Gag-specific tetramer. In addition, at euthanasia, antigen-specific cells producing gamma interferon or tumor necrosis factor alpha from the jejunum lamina propria were quantified in all macaques. Surprisingly, antigen-specific CD8+ T cells were found in the mucosal tissues of all immunized macaques regardless of whether the vaccine was administered by a mucosal route (intranasal or intrarectal) or systemically. In addition, following mucosal SIVmac251 challenge, antigen-specific responses were mainly confined to mucosal tissues, again regardless of the route of immunization. We conclude that immunization with a live vector vaccine results in the appearance of CD8+ T-cell responses at mucosal sites even when the vaccine is delivered by nonmucosal routes.