Elizabeth Ann L. Enninga

Fetal Sex-Based Differences in Maternal Hormones, Angiogenic Factors, and Immune Mediators During Pregnancy and the Postpartum Period

Several pregnancy complications have disparities based on the sex of the fetus. It is unknown whether the sex of the fetus differentially alters the maternal immune milieu, potentially contributing to the observed differences.

Growth Modeling of the Maternal Cytokine Milieu throughout Normal Pregnancy: Macrophage-Derived Chemokine Decreases as Inflammation/Counterregulation Increases

Several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy, but most studies have been cross-sectional or of relatively few time points. Levels of 42 cytokines were determined using a multiplex bead-based assay on archived serum from a cohort of pregnant women (N = 16) at median of 18 time points tested, from the first trimester through to parturition, per woman. Unconditional growth modeling was then used to determine time-dependent changes in levels of these cytokines. Macrophage-derived chemokine (MDC, aka CCL22) decreases as pregnancy progresses. IL-1β, IL-6, IL-8, IL-12p70, IL-13, IL-15, IP-10, and FLT3-ligand increase as a function of gestational weeks, and IFNα2, IL-1ra, IL-3, IL-9, IL-12p40, and soluble CD40 ligand increase as a function of trimester. As pregnancy normally progresses, a maternal shift away from a type 2-biased immune response and toward an inflammatory/counterregulatory response is observed.

Galectin-9 modulates immunity by promoting Th2/M2 differentiation and impacts survival in patients with metastatic melanoma.

Galectin-9, a &bgr;-galactoside-binding protein, is defined as a negative regulator of T helper 1 (Th1) immune responses, favoring Th2 bias. Systemic immunity in patients with metastatic melanoma is predominantly Th2 biased. We hypothesized that galectin-9 can modulate systemic immunity toward Th2 polarization in patients with advanced melanoma. The presence or concentration of galectin-9 was assessed in tumors and plasma, in patients with metastatic melanoma. The immunomodulatory function of galectin-9 was determined by exposing human peripheral blood mononuclear cells to galectin-9 in vitro. Galectin-9 was expressed in 57% of tumors and was significantly (3.6-fold) increased in the plasma of patients with advanced melanoma compared with healthy controls (P<0.001). High plasma galectin-9 concentration was associated with systemic Th2 polarization and reduced 2-year survival compared with low/no galectin-9 expression. In-vitro, galectin-9 reduced proliferation of healthy peripheral blood mononuclear cells, and promoted Th1 cell apoptosis, Th2-biased cell phenotypes, and cytokine secretion. Galectin-9 also stimulated monocyte differentiation toward an M2 macrophage phenotype, as assessed by chemokine/cytokine secretion and CD206 expression, observed both in vitro as well as in patients with metastatic melanoma. Elevated galectin-9 in patient plasma correlated with Th2 systemic bias and less favorable clinical outcomes for patients with metastatic melanoma. This Th2 bias appears to be not only a feature of the known mechanisms of Th1 apoptosis by T-cell immunoglobulin and mucin-domain containing-3 binding, but also mediated by myeloid cell differentiation toward an M2 phenotype, that favors tumor progression. These data support galectin-9 as a novel therapeutic target for patients with metastatic melanoma.

Evidence of Th2 polarization of the sentinel lymph node (SLN) in melanoma

Melanoma has a propensity for lymphatogenous metastasis. Improved understanding of the sentinel lymph node (SLN) immunological environment may improve outcomes. The immune phenotype of fresh melanoma SLNs (n = 13) were compared to fresh control lymph nodes (n = 13) using flow cytometry. RNA was isolated from CD4+ T cells of the SLN and control lymph node and assessed for Th1/Th2 gene expression pathways using qRT-PCR. In addition, VEGF expression was compared between primary melanoma (n = 6) and benign nevi (n = 6) using immunohistochemistry. Melanoma SLNs had fewer CD8+ T cells compared to controls (9.2% vs. 19.5%, p = 0.0005). The CD8+ T cells within the SLN appeared to have an exhausted phenotype demonstrated by increased PD-1 mRNA expression (2.2% vs. 0.8%, p = 0.004) and a five-fold increase in CTLA-4 mRNA expression. The SLN also contained an increased number of CD14 (22.7% vs. 7.7%, p = 0.009) and CD68 (9.3% vs. 2.7%, p = 0.001) macrophages, and CD20 B cells (31.1% vs. 20.7%, p = 0.008), suggesting chronic inflammation. RT-PCR demonstrated a significant Th2 bias within the SLN. In vitro studies demonstrated a similar Th2 polarization with VEGF treatment of control lymph nodes. The primary melanoma demonstrated strong VEGF expression and an increase in VEGFR1 within the SLN. Melanoma is associated with Th2-mediated “chronic inflammation,” fewer cytotoxic T cells, and an exhausted T cell phenotype within the SLN combined with VEGF overproduction by the primary melanoma. These immunologic changes precede nodal metastasis and suggests consideration of VEGF inhibitors in future immunotherapy studies.

Survival of cutaneous melanoma based on sex, age, and stage in the United States, 1992–2011

Women diagnosed with cutaneous melanoma have a survival advantage compared to men, which has been hypothesized to be due to difference in behavior and/or biology (sex hormones). It remains controversial whether this advantage is dependent on age or stage of disease. We sought to compare melanoma‐specific survival between females in pre, peri, and postmenopausal age groups to males in the same age group, adjusting for stage of disease. This is a retrospective population‐based cohort study using the Surveillance, Epidemiology, and End Results (SEER) database. Patients diagnosed from 1 January 1992 through 31 January 2011 with primary invasive cutaneous melanoma were included in our cohort. Melanoma‐specific survival was the main outcome studied. Of the 106,511 subjects that were included, 45% were female. Females in all age groups (18–45, 46–54, and ≥55) with localized and regional disease, were less likely to die from melanoma compared to males in the same age group. Among patients with localized and regional disease, the relative risk of death due to melanoma increased with advancing age at diagnosis; this increase was more pronounced among females than males. In contrast, we observed no female survival advantage among patients with distant disease and no effect of age on relative risk of death from melanoma. Females with localized and regional melanoma have a decreased risk of death compared to males within all age groups. Our data show no differences in survival between men and women with metastatic melanoma, indicating that the influence of sex on survival is limited to early stage disease but not confined to pre or perimenopausal age groups.

Immune Reactivation by Cell-Free Fetal DNA in Healthy Pregnancies Re-Purposed to Target Tumors: Novel Checkpoint Inhibition in Cancer Therapeutics.

The role of the immune system in cancer progression has become increasingly evident over the past decade. Chronic inflammation in the promotion of tumorigenesis is well established, and cancer-associated tolerance/immune evasion has long been appreciated. Recent developments of immunotherapies targeting cancer-associated inflammation and immune tolerance, such as cancer vaccines, cell therapies, neutralizing antibodies, and immune checkpoint inhibitors, have shown promising clinical results. However, despite significant therapeutic advances, most patients diagnosed with metastatic cancer still succumb to their malignancy. Treatments are often toxic, and the financial burden of novel therapies is significant. Thus, new methods for utilizing similar biological systems to compare complex biological processes can give us new hypotheses for combating cancer. One such approach is comparing trophoblastic growth and regulation to tumor invasion and immune escape. Novel concepts regarding immune activation in pregnancy, especially reactivation of the immune system at labor through toll like receptor engagement by fetal derived DNA, may be applicable to cancer immunotherapy. This review summarizes mechanisms of inflammation in cancer, current immunotherapies used in the clinic, and suggestions for looking beyond oncology for novel methods to reverse cancer-associated tolerance and immunologic exhaustion utilizing mechanisms encountered in normal human pregnancy.

CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment: Galectin-9/CD206 binding

In patients with metastatic melanoma, high blood levels of galectin‐9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin‐9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin‐9 with macrophages. We aimed to determine whether galectin‐9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor‐supportive. It was determined that incubation of CD68+ macrophages with galectin‐9 or anti‐CD206 blocked target binding and that both CD206 and galectin‐9 were detected by immunoprecipitation of cell lysates. CD206 and galectin‐9 had a binding affinity of 2.8 × 10−7 m. Galectin‐9 causes CD206+ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP‐1), but less macrophage‐derived chemokine (MDC). Galectin‐9 had no effect on classical monocyte subsets, but caused expansion of the non‐classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin‐9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin‐9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti‐tumor effects by macrophages. Copyright

Immune checkpoint molecules soluble program death ligand 1 and galectin-9 are increased in pregnancy

Pregnancy requires balance between tolerance to the haploidentical fetus and the mothers ability to mount immune responses. There are parallels to this phenomenon that occur in metastatic cancer. We assessed soluble program death ligand‐1 soluble PD‐L1 (sPD‐L1) and galectin‐9 in the blood of pregnant women during gestation as these molecules are highly involved in immune suppression during cancer.

MIHALAS et al.The Primordial Journey.

Development and differentiation of primordial germ cells (PGCs) marks the origin of the gamete and is essential for sexual reproduction. During mouse embryogenesis, PGCs migrate from the base of the allantois through the embryonic hindgut to colonize the gonadal ridge and undergo mitotic proliferation. This article is protected by copyright. All rights reserved.

Abstract A41: Galectin-9 has tumor promoting properties that may be targetable in metastatic melanoma

Introduction: Galectin-9 (Gal-9) is a carbohydrate-binding protein that interacts with T-cell immunoglobulin mucin 3 (TIM3) to modulate immune homeostasis. The Gal-9/TIM3 interaction down regulates activated T cells which promotes tumor growth, much like program death 1 (PD-1) and its ligand (PD-L1). Still, the effect Gal-9 has on the immune environment of melanoma patients is weakly understood. This study focuses on the role of Gal-9 in immunoregulation of healthy persons and metastatic melanoma patients. Methods: Soluble Gal-9 levels were determined from plasma collected from patients and healthy persons using standard ELISA methods. Proliferation and apoptosis of cells in the presence and absence of Gal-9 was determined by mixed lymphocyte reactions and Tunnel staining. Supernatants from these reactions were collected and run using a cytokine bead assay to determine changes in immune parameters with and without Gal-9. Results: Soluble Gal-9 was significantly higher in metastatic melanoma patients compared to healthy controls (3929 vs. 877 pg/mL, p=0.0003 respectively). In the presence of Gal-9, the proliferative effect of an allostimulus could be overridden until the level of responder cells to stimulator cells was 4:1. Using an apoptosis assay, Gal-9 was found to increase the level of cellular apoptosis; however, apoptosis was decreased as the allostimulus increased. Healthy cells incubated with Gal-9 were found to increase secretion of growth factors (EGF and FGF-2), chemokines (CXCL1, CXCL8, CCL2 and CCL3), T helper 1 cytokines (IFN-g and IL-7) which have shown tumor promoting affects and T helper 2 cytokines (IL-13 and IL-4). Conclusions: Gal-9 may potentially play a role in suppressing anti-tumor immunity. This suggests the use of a Gal-9 neutralizing antibody for therapeutic benefit in melanoma. Citation Format: Elizabeth Ann L. Enninga, Wendy K. Nevala, Svetomir N. Markovic. Galectin-9 has tumor promoting properties that may be targetable in metastatic melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A41.