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We consider the Dirichlet problem for second-order linear elliptic equations in divergence form − d i v ( A ∇ u ) + b ⋅ ∇ u + λ u = f + d i v F in Ω and u = 0 on ∂ Ω , \begin{equation*} -div(A\nabla u)+\mathbf {b} \cdot \nabla u+\lambda u=f+div\mathbf {F}\quad \text {in } \Omega \quad \text {and}\quad u=0\quad \text {on } \partial \Omega , \end{equation*} in bounded Lipschitz domain Ω \Omega in R 2 \mathbb {R}^2 , where A : R 2 → R 2 2 A:\mathbb {R}^2\rightarrow \mathbb {R}^{2^2} , b : Ω → R 2 \mathbf {b} : \Omega \rightarrow \mathbb {R}^2 , and λ ≥ 0 \lambda \geq 0 are given. If 2 > p > ∞ 2>p>\infty and A A has a small mean oscillation in small balls, Ω \Omega has small Lipschitz constant, and d i v A , b ∈ L 2 ( Ω ; R 2 ) divA,\,\mathbf {b} \in L^{2}(\Omega ;\mathbb {R}^2) , then we prove existence and uniqueness of weak solutions in W 0 1 , p ( Ω ) W^{1,p}_0(\Omega ) of the problem. Similar result also holds for the dual problem.

Mild Systemic Autoimmunity in Mice with GARP-/- Treg Cells

Activated regulatory T (Treg) cells express the surface receptor glycoprotein-A repetitions predominant (GARP), which binds and activates latent TGFβ. How GARP modulates Treg function in inflammation and cancer remains unclear. Here we demonstrate that loss of GARP in Treg cells leads to spontaneous inflammation with highly activated CD4+ and CD8+ T cells and development of enteritis. Treg cells lacking GARP were unable to suppress pathogenic T-cell responses in multiple models of inflammation, including T-cell transfer colitis. GARP−/− Treg cells were significantly reduced in the gut and exhibited a reduction in CD103 expression, a colon-specific migratory marker. In the colitis-associated colon cancer model, GARP on Treg cells dampened immune surveillance, and mice with GARP−/− Treg cells exhibited improved antitumor immunity. Thus, GARP empowers the functionality of Treg cells and their tissue-specific accumulation, highlighting the importance of cell surface TGFβ in Treg function and GARP as a potential therapeutic target for colorectal cancer therapy.Significance: These findings uncover functions of membrane-bound TGFβ and GARP that tune the activity of Treg cells, highlighting a potential treatment strategy in autoimmune diseases and cancer.

Immune checkpoint blockade (ICB) has revolutionized cancer immunotherapy. However, most patients with cancer fail to respond clinically. One potential reason is the accumulation of immunosuppressive transforming growth factor β (TGFβ) in the tumor microenvironment (TME). TGFβ drives cancer immune evasion in part by inducing regulatory T cells (Tregs) and limiting CD8We investigated the role of GARP in human patients with cancer by analyzing existing large databases. In addition, we generated and humanized an anti-GARP monoclonal antibody and evaluated its antitumor efficacy and underlying mechanisms of action in murine models of cancer.We demonstrate that GARP overexpression in human cancers correlates with a tolerogenic TME and poor clinical response to ICB, suggesting GARP blockade may improve cancer immunotherapy. We report on a unique anti-human GARP antibody (named PIIO-1) that specifically binds the ligand-interacting domain of all latent TGFβ isoforms. PIIO-1 lacks recognition of GARP-TGFβ complex on platelets. Using humanGARP contributes to multiple aspects of immune resistance in cancer. Anti-human GARP antibody PIIO-1 is an efficacious and safe strategy to block GARP-mediated LTGFβ activation, enhance CD8

Sex bias exists in the development and progression of nonreproductive organ cancers, but the underlying mechanisms are enigmatic. Studies so far have focused largely on sexual dimorphisms in cancer biology and socioeconomic factors. Here, we establish a role for CD8 + T cell–dependent antitumor immunity in mediating sex differences in tumor aggressiveness, which is driven by the gonadal androgen but not sex chromosomes. A male bias exists in the frequency of intratumoral antigen-experienced Tcf7 /TCF1 + progenitor exhausted CD8 + T cells that are devoid of effector activity as a consequence of intrinsic androgen receptor (AR) function. Mechanistically, we identify a novel sex-specific regulon in progenitor exhausted CD8 + T cells and a pertinent contribution from AR as a direct transcriptional transactivator of Tcf7 /TCF1. The T cell–intrinsic function of AR in promoting CD8 + T cell exhaustion in vivo was established using multiple approaches including loss-of-function studies with CD8-specific Ar knockout mice. Moreover, ablation of the androgen-AR axis rewires the tumor microenvironment to favor effector T cell differentiation and potentiates the efficacy of anti–PD-1 immune checkpoint blockade. Collectively, our findings highlight androgen-mediated promotion of CD8 + T cell dysfunction in cancer and imply broader opportunities for therapeutic development from understanding sex disparities in health and disease.

INTRODUCTION AND OBJECTIVE:Recently single-cell RNA sequencing (scRNA-Seq) has increased our depth of understanding of genomic features of distinct cellular populations. To reveal the dynamics of b...

Background: Remarkable discrepancy exists in outcomes between men and women for multiple malignancies. We sought to expose sex differences in using platelet count and neutrophil-to-lymphocyte ratio (NLR) to predict overall survival for select cancer types with focus on head and neck squamous cell carcinoma (HNSCC). Methods: Peripheral blood samples from 9,365 patients seen in a tertiary teaching hospital with nine different primary tumors were retrospectively examined. HNSCC RNA-sequencing data from The Cancer Genome Atlas were analyzed by two computational means [Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE)] to extend our observations to the tumor microenvironment. Results: For HNSCC, platelet count was more predictive of overall survival for males [log-rank test: HR = 1.809; 95% confidence interval (CI), 1.461–2.239 vs. HR = 1.287; 95% CI, 0.8901–1.861], whereas NLR was more predictive for females (HR = 2.627; 95% CI, 1.716–4.02 vs. HR = 1.261; 95% CI, 0.998–1.593). For females, lymphocyte count was more associated with survival than neutrophil count (multivariate Cox regression: P = 0.0015 vs. P = 0.7476). Both CIBERSORT (P = 0.0061) and ESTIMATE (P = 0.022) revealed greater immune infiltration in females. High tumor infiltration by T lymphocytes was more strikingly associated with survival in females (HR = 0.20, P = 0.0281) than in males (HR = 0.49, P = 0.0147). Conclusions: This is the first study to comprehensively demonstrate sex bias in the clinical utility of platelet, granulocyte, and lymphocyte counts as biomarkers to prognosticate HNSCC patients. Impact: This work emphasizes the necessity to consider sex in appraising inflammatory markers for cancer risk stratification. Cancer Epidemiol Biomarkers Prev; 27(10); 1176–85. ©2018 AACR.

Men and women show striking yet unexplained discrepancies in incidence, clinical presentation, and therapeutic response across different types of infectious/autoimmune diseases and malignancies1,2. For instance, bladder cancer shows a 4-fold male-biased incidence that persists after adjustment for known risk factors3,4. Here, we utilize murine bladder cancer models to establish that male-biased tumor burden is driven by sex differences in endogenous T cell immunity. Notably, sex differences exist in early fate decisions by intratumoral CD8+ T cells following their activation. While female CD8+ T cells retain their effector function, male counterparts readily adopt a Tcf1lowTim3− progenitor state that becomes exhausted over tumor progression. Human cancers show an analogous male-biased frequency of exhausted CD8+ T cells. Mechanistically, we describe an opposing interplay between CD8+ T cell intrinsic androgen and type I interferon5,6 signaling in Tcf1/Tcf7 regulation and formation of the progenitor exhausted T cell subset. Consistent with female-biased interferon response7, testosterone-dependent stimulation of Tcf1/Tcf7 and resistance to interferon occurs to a greater magnitude in male CD8+ T cells. Male-biased predisposition for CD8+ T cell exhaustion suggests that spontaneous rejection of early immunogenic bladder tumors is less common in males and carries implications for therapeutic efficacy of immune checkpoint inhibitors8,9.

We consider the following Dirichlet problems for elliptic equations with singular drift b: (a) − div ( A ∇ u ) + div ( u b ) = f , (b) − div ( A T ∇ v ) − b ⋅ ∇ v = g in Ω , where Ω is a bounded Lipschitz domain in R n , n ≥ 2 . Assuming that b ∈ L n , ∞ ( Ω ) n has non-negative weak divergence in Ω, we establish existence and uniqueness of weak solution in W 0 1 , 2 + e ( Ω ) of the problem (b) when A is bounded and uniformly elliptic. As an application, we prove existence and uniqueness of weak solution u in ⋂ q 2 W 0 1 , q ( Ω ) for the problem (a) for every f ∈ ⋂ q 2 W − 1 , q ( Ω ) .

Activated regulatory T (Treg) cells express the surface receptor glycoprotein-A repetitions predominant (GARP), which binds and activates latent TGFβ. How GARP modulates Treg function in inflammation and cancer remains unclear. Here we demonstrate that loss of GARP in Treg cells leads to spontaneous inflammation with highly activated CD4+ and CD8+ T cells and development of enteritis. Treg cells lacking GARP were unable to suppress pathogenic T-cell responses in multiple models of inflammation, including T-cell transfer colitis. GARP−/− Treg cells were significantly reduced in the gut and exhibited a reduction in CD103 expression, a colon-specific migratory marker. In the colitis-associated colon cancer model, GARP on Treg cells dampened immune surveillance, and mice with GARP−/− Treg cells exhibited improved antitumor immunity. Thus, GARP empowers the functionality of Treg cells and their tissue-specific accumulation, highlighting the importance of cell surface TGFβ in Treg function and GARP as a potential therapeutic target for colorectal cancer therapy. Significance: These findings uncover functions of membrane-bound TGFβ and GARP that tune the activity of Treg cells, highlighting a potential treatment strategy in autoimmune diseases and cancer.

Gp96 is an essential endoplasmic reticulum chaperone for various proteins including GARP, TLRs, integrins, Wnt co-receptor LRP6 and the platelet glycoprotein Ib/IX/V complex. Our laboratory has previously shown that loss of gp96 in murine Foxp3+ regulatory T cells (Tregs) results in their instability as determined by FOXP3 downregulation, impaired immunosuppressive function and production of effector cytokines (Zhang, et al., J Clin Invest., 2015, 125: 859–869). However, the mechanism underlying Treg dysfunction upon gp96 deletion remains unknown. Using tamoxifen-inducible Treg-specific gp96 gene (encoded by Hsp90b1) knockout (KO) mice (Foxp3-ERT2creHsp90b1f/f), we first phenotypically characterized gp96-deleted Tregs in mouse spleen and peripheral lymph nodes (pLNs). Gp96 deficient Tregs showed decreased frequency of TCF1−CD62L− effector population, but were enriched with short-lived KLRG1+ and Helios+ subsets, in both spleen and pLNs. We also confirmed that the expression of Foxp3 was decreased in splenic gp96-deleted Tregs, which correlated with a reduction of cell surface CD25. Intriguingly, both Foxp3 and surface CD25 levels stayed high in gp96-null Tregs in pLNs. In addition, gp96-null Tregs in mouse spleen, but not in pLNs, demonstrated compromised suppressive function. We further found that IL-2/CD25 signaling, as assessed by STAT5 phosphorylation, was more activated in gp96-null Tregs in pLNs, compared to that in spleen. In vitro IL-2 treatment was able to rescue the attenuated Foxp3 expression in gp96-null Tregs. Our work indicates that IL-2/pSTAT-5 signaling may be a key factor in maintaining Treg Foxp3 expression and its lineage stability upon gp96 deletion.

559 Background: Myeloid immune cells such as myeloid derived suppressor cells (MDSC) and tumor associated macrophages (TAM) have been hypothesized to cause resistance to immune checkpoint blockade (ICB). This is a pressing clinical problem for patients with bladder cancer. Here we determined if we could identify immune cells associated with resistance to ICB in the BBN963 mouse model, and if we could identify therapeutic strategies to target those same suppressor immune cells from patients with bladder cancer. Methods: BBN963 subcutaneous allografts were established in C57BL6/J mice. Response to anti-PD-L1 ICB was classified as partial or complete response according to RECIST criteria. Immune cell subsets with the tumors was evaluated by gene expression profiling and flow cytometry. Peripheral blood from patients with bladder cancer was collected under an IRB-approved protocol. MDSC were purified by flow sorting (CD11b+ CD33+ HLA-DRlow/neg) and screened for viability (Annexin-V staining) after 24 hours of exposure to a panel potential MDSC inhibitors. Results: 16/22 (72%) subjects met criteria for partial or complete response, while 6/22 (28%) were classified as anti-PD-L1 non-responders. Mice in the control group had a 0/10 (0%) response to isotype control (IgG) treatment. Monocytic MDSC (CD11b+ Ly6C+) were much more frequent among the intratumoral CD45+ cells of non-responding subjects as compared to control mice. Nanostring immune panel gene expression profiling revealed that combination treatment of tumor bearing mice with anti-PD-L1 plus ibrutinib (a putative MDSC inhibitor) decreased mRNA biomarkers of tumor-infiltrating macrophages. In vitro screening of patient-derived peripheral blood mononuclear cells showed that an inhibitor of the bromodomain and extraterminal domain (BET) family BRD4 specifically decreased MDSC viability. Conclusions: Monocytic MDSC appear to be associated with resistance to anti-PD-L1 ICB in a new murine model. Analysis of MDSC from patients with bladder cancer suggests that these myeloid suppressor cells can be specifically targeted.

Aspergillus flavus and A. parsiticus produce aflatoxins that are highly toxic to mammals and birds. In this study, the inhibitory effects of 1,8-cineole and t-cinnamaldehyde were examined on the growth of Aspergillus flavus ATCC 22546 and aflatoxin production. 1,8-Cineole showed 50% inhibition of fungal growth at a concentration of 250 ppm, while t-cinnamaldehyde almost completely inhibited fungal growth at a concentration of 50 ppm. Furthermore, no fungal growth was observed when the growth medium was treated with 100 ppm t-cinnamaldehyde. 1,8-Cineole also exhibited 50% inhibition on the production of aflatoxin B1 and aflatoxin B2 at a concentration of 100 ppm, while the addition of 100 ppm t-cinnamaldehyde completely inhibited aflatoxin production. These antiaflatoxigenic activities were related to a dramatic downregulation of the expression of aflE and aflL by 1,8-cineole, but the mode of action for t-cinnamaldehyde was unclear. Collectively, our results suggest that both of the compounds are promising alternatives to the currently used disinfectant, propionic acid, for food and feedstuff preservation.

We consider the Dirichlet and Neumann problems for second-order linear elliptic equations: \[ -\triangle u +\mathrm{div}(u\mathbf{b}) =f \quad\text{ and }\quad -\triangle v -\mathbf{b} \cdot \nabla v =g \] in a bounded Lipschitz domain $\Omega$ in $\mathbb{R}^n$ $(n\geq 3)$, where $\mathbf{b}:\Omega \rightarrow \mathbb{R}^n$ is a given vector field. Under the assumption that $\mathbf{b} \in L^{n}(\Omega)^n$, we first establish existence and uniqueness of solutions in $L_{\alpha}^{p}(\Omega)$ for the Dirichlet and Neumann problems. Here $L_{\alpha}^{p}(\Omega)$ denotes the Sobolev space (or Bessel potential space) with the pair $(\alpha,p)$ satisfying certain conditions. These results extend the classical works of Jerison-Kenig [17] and Fabes-Mendez-Mitrea [12] for the Poisson equation. We also prove existence and uniqueness of solutions of the Dirichlet problem with boundary data in $L^{2}(\partial\Omega)$. Our results for the Dirichlet problems hold even for the case $n=2$., Comment: 38 pages, 7 figures

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymerization factors, such as formins, may participate in protrusion formation. Here, we show that Arp2/3 localized to only a minor portion of the protrusion. Moreover, treatment of L. monocytogenes-infected HeLa cells with a formin FH2-domain inhibitor significantly reduced protrusion length. In addition, the Diaphanous-related formins 1-3 (mDia1-3) localized to protrusions, and knockdown of mDia1, mDia2, and mDia3 substantially decreased cell-to-cell spread of L. monocytogenes. Rho GTPases are known to be involved in formin activation. Our studies also show that knockdown of several Rho family members significantly influenced bacterial cell-to-cell spread. Collectively, these findings identify a Rho GTPase-formin network that is critically involved in the cell-to-cell spread of L. monocytogenes.

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3–5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3–5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

Cluster of differentiation 47 (CD47) (also known as integrin-associated protein) is a ubiquitously expressed glycoprotein of the immunoglobulin superfamily that plays a critical role in self-recognition. Various solid and hematologic cancers exploit CD47 expression in order to evade immunological eradication, and its overexpression is clinically correlated with poor prognoses. One essential mechanism behind CD47-mediated immune evasion is that it can interact with signal regulatory protein-alpha (SIRPα) expressed on myeloid cells, causing phosphorylation of the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and recruitment of Src homology 2 domain-containing tyrosine phosphatases to ultimately result in delivering an anti-phagocytic—“don’t eat me”—signal. Given its essential role as a negative checkpoint for innate immunity and subsequent adaptive immunity, CD47-SIRPα axis has been explored as a new target for cancer immunotherapy and its disruption has demonstrated great therapeutic promise. Indeed, CD47 blocking antibodies have been found to decrease primary tumor size and/or metastasis in various pre-clinical models. In this review, we highlight the various functions of CD47, discuss anti-tumor responses generated by both the innate and adaptive immune systems as a consequence of administering anti-CD47 blocking antibody, and finally elaborate on the clinical potential of CD47 blockade. We argue that CD47 is a checkpoint molecule for both innate and adaptive immunity for tumor evasion and is thus a promising target for cancer immunotherapy.