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In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4+ T cells, whereas numbers of circulating CD8+ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

The stress-inducible chaperone protein HSP70 (HSPA1) is implicated in melanoma development, and HSP70 inhibitors exert tumor-specific cytotoxic activity in cancer. In this study, we documented that a significant proportion of melanoma tumors express high levels of HSP70, particularly at advanced stages, and that phospho-FAK (PTK2) and BRAF are HSP70 client proteins. Treatment of melanoma cells with HSP70 inhibitors decreased levels of phospho-FAK along with impaired migration, invasion, and metastasis in vitro and in vivo. Moreover, the HSP70 inhibitor PET-16 reduced levels of mutant BRAF, synergized with the BRAF inhibitor PLX4032 in vitro, and enhanced the durability of response to BRAF inhibition in vivo. Collectively, these findings provide strong support for HSP70 inhibition as a therapeutic strategy in melanoma, especially as an adjuvant approach for overcoming the resistance to BRAF inhibitors frequently observed in melanoma patients. Cancer Res; 76(9); 2720–30. ©2016 AACR.

Identification of Rb1 induction in chronic asymptomatic HIV-1 infection as a mediator of apoptosis resistance in monocytes, in association with protective autophagy. We have previously described an antiapoptotic steady-state gene expression profile in circulating human monocytes from asymptomatic viremic HIV+ donors, but the mechanism associated with this apoptosis resistance remains to be fully elucidated. Here, we show that Rb1 activation is a dominant feature of apoptosis resistance in monocytes exposed to HIV-1 in vivo (as measured ex vivo) and in vitro. Monocytes from asymptomatic viremic HIV+ individuals show a positive correlation between levels of hypophosphorylated (active) Rb1 and VL in conjunction with increases in other p53-inducible proteins associated with antiapoptosis regulation, such as p21 and PAI-1 (SERPINE1), when compared with circulating monocytes from uninfected donors. Monocytes exposed in vitro to HIV-1 R5 isolates but not X4 isolates showed lower caspase-3 activation after apoptosis induction, indicating a role for the CCR5 signaling pathway. Moreover, monocytes exposed to R5 HIV-1 or MIP-1β induced Rb1 and p21 expression and an accumulation of autophagy markers, LC3 and Beclin. The inhibition of Rb1 activity in HIV-1 R5 viral-exposed monocytes using siRNA led to increased apoptosis sensitivity, thereby confirming a central role for Rb1 in the antiapoptotic phenotype. Our data identify Rb1 induction in chronic asymptomatic HIV-1 infection as a mediator of apoptosis resistance in monocytes in association with protective autophagy and contributing to monocyte survival during immune activation and/or HIV-1 viremia.

Oncogenes often upregulate nutrient sensing and utilizing pathways, leading to altered metabolism in tumor cells. RAS-driven tumors typify cancers undergoing marked metabolic reprogramming. In melanoma, RAS signaling is often deregulated; while activating mutations in NRAS are present in >25% of tumors, RAS-driven tumors account for almost 50% of cases. However, there are no effective therapies for this type of tumors. Although oncogenic NRAS activates the MAPK pathway, inhibition of this pathway alone has limited antitumor efficacy and most NRAS-mutant tumors are resistant to MAPK inhibitor (MAPKi) monotherapy. We discovered that NRAS-mutant melanomas resistant to MAPKi are highly dependent on the ribosomal protein S6 kinase 2 (S6K2). We demonstrate that loss of S6K2, a signaling effector of the mTORC1 nutrient-sensing pathway, triggers cell death selectively in NRAS-mutant melanoma resistant to MAPKi. Acute depletion of S6K2 enhanced ROS production, lipid synthesis and accumulation of intracellular unsaturated fatty acids. ROS susceptible (poly)-unsaturated fatty acids sensitized cells to ROS, resulting in lipid peroxidation and oxidative cell death. We further determined that S6K2 depletion was coupled to increased expression of markers of apoptosis and ferroptosis, suggesting that S6K2 blockade could trigger multiple forms of cell death, including apoptotic and a ferroptotic-like cell death. Notably, co-inhibition of S6K1 (a S6K isoform that is co-regulated by mTORC1) together with S6K2 diminished the effects of S6K2 blockade, suggesting that selective inhibition of S6K2 is required to induce cell death. Indeed, while silencing of S6K2 triggered lipid peroxidation and oxidative cell death and S6K1 silencing had negligible effects, concomitant depletion of S6K1 and S6K2 attenuated lipid peroxidation and cell death. Mimicking the effects of S6K2 loss by combining an ROS-inducing agent with unsaturated fatty acids or treating tumor cells with lipid peroxidation inducers restrained NRAS-mutant melanoma growth in vitro and in vivo. Taken together, our studies have identified a critical vulnerability of NRAS-mutant melanoma by uncoupling S6K1 and S6K2 to trigger metabolic dysfunction and tumor cell death. This strategy is remarkably distinct from using PI3K/mTOR inhibitors, which concurrently suppress S6K1 and S6K2 and are often cytostatic. We propose that this paradigm could be exploited to develop therapeutic approaches targeting NRAS-mutant tumors and overcome resistance to MAPKi in the context of oncogenic NRAS. Citation Format: Hsin-Yi Chen, Minu Samanta, Patricia Reyes-Uribe, Andrew V. Kossenkov, Xiangfan Yin, Yiling Lu, Mark Axelrod, Michael J. Weber, Qin Liu, Gordon B. Mills, Jessie Villanueva. Loss of S6K2 perturbs redox balance and fatty acid metabolism, leading to oxidative cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2381.

Sex workers practicing in high HIV endemic areas have been extensively targeted to test anti-HIV prophylactic strategies. We hypothesize that in women with high levels of genital exposure to semen changes in cervico-vaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity and immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers (FSWs; n=50), as compared with control women (CG; n=32). FSWs had low-to-absent HIV-1-specific immune responses with significantly lower CD38 expression on circulating CD4(+) or CD8(+) T-cells (both: P

An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance. Significance: These data show for the first time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib. Cancer Discov; 3(12); 1378–93. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1317

Nature 532, 250–254 (2016); doi:10.1038/nature17392 In Fig. 5a of this Letter, the labels PBS and PLX4720 were inadvertently reversed. The corrected Fig. 5a is shown in Fig. 1 of this Corrigendum.

Persistence of human papillomavirus (HPV) and cervical disease in the context of HIV co-infection can be influenced by introduction of antiretroviral therapy (ART) and sustained immune activation despite ART. We conducted a cross-sectional study in order to evaluate immune activation/exhaustion in ART-suppressed HIV(+) women with or without high-risk (HR) HPV-related cervical intraepithelial neoplasia (CIN). 55 South African women were recruited in three groups: HR (-) (n = 16) and HR (+) (n = 15) HPV with negative cervical histopathology, and HR (+) HPV with CIN grade 1/2/3 (n = 24). Sampling included endocervical brushing (HPV DNA genotyping), Pap smear (cytology), colposcopic punch biopsy (histopathology, histochemical evaluation of immune cells), and peripheral blood (clinical assessment, flow cytometry-based immune subset characterization). Statistics were done using R2.5.1. Irrespective of the presence of CIN, HR (+) HPV women had higher circulating levels of T cells expressing markers of activation/exhaustion (CD38, PD1, CTLA-4, BTLA, CD160), Tregs, and myeloid subsets expressing corresponding ligands (PDL1, PDL2, CD86, CD40, HVEM) than HR (-) HPV women. A decrease in circulating NK cells was associated with CIN grade. CD4(+) T cell count associated negatively with T cell exhaustion and expression of negative regulators on myeloid cells. Women with CIN when compared to HR (-) HPV women, had higher cervical cell density in stroma and epithelium for CD4(+), CD68(+), and CD11c(+) cells, and only in stroma for CD8(+) cells. We conclude that in ART-suppressed HIV-infected women with HPV co-infection the levels of T and myeloid cell activation/exhaustion are associated with the presence of HR HPV genotypes.

Objective: Sustained antiretroviral therapy (ART)-mediated viral suppression restores responses to vaccination in HIV-1-infected individuals. As ART interruption occur frequently in resource-constrained settings, we studied their effects on the ability to mount humoral immune responses against a neoantigen. Design: Treatment-naive HIV-1-infected individuals were treated with stavudine, lamuvidine and lopinavir/ritonovir. Individuals who maintained viral load less than 50copies/ml and CD4 þ T-cell counts more than 450cells/ml for 6 months received three doses of rabies vaccine, and were randomized to 72 weeks of continuous ART (arm 1) or sequential 2, 4 and 8-week ART interruptions (arm 2). An additional vaccine dose was administered at study end. Methods: Neutralizing antibody titers to rabies virus were assessed in plasma with a rapid fluorescent focus-inhibiting test. Results: The proportion of participants achieving protective (>0.5IU/ml) antibody titer after vaccination was similar (arm 1 ¼92%; arm 2 ¼91%), but over time the cumulative proportion of observations with protective titer was greater in arm 1 than arm 2 (P ¼0.0177). From week 26 after vaccination, antibody titers were lower in arm 2 than arm 1, and volunteers in arm 2 lost protective antibody titers at a greater rate (P ¼0.0029). After boosting, 100% of arm 1 and 95% arm 2 volunteers achieved protective antibody titer. Conclusion: Our data indicate that individuals undergoing recurring ART interruption retain lower neutralizing antibody titers to a neoantigen, but maintain the ability to mount secondary responses upon boosting, suggesting that they might benefit from vaccine schedule intensification. 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2012, 26:1355‐1362

The effect of early versus deferred antiretroviral treatment (ART) on plasma concentration of lipopolysaccharide (LPS) and host LPS-binding molecules in human immunodeficiency virus (HIV)-infected infants up to 1 year of age was investigated.We evaluated 54 perinatally HIV-infected and 22 HIV-exposed uninfected infants (controls) at the first and second semester of life. All HIV-infected infants had a baseline CD4 of ≥ 25%, participated in the Comprehensive International Program of Research on AIDS Children with HIV Early Antiretroviral Therapy trial in South Africa, and were randomized in the following groups: group 1 (n = 20), ART deferred until CD425% or severe HIV disease; and group 2 (n = 34), ART initiation within 6 to 12 weeks of age. LPS, endotoxin-core antibodies, soluble CD14 (sCD14), and LPS-binding protein (LBP) were measured in cryopreserved plasma. T-cell activation was measured in fresh whole blood.At the first semester, LPS concentration was higher in HIV-infected infants than in controls; sCD14, LBP, and T-cell activation were higher in group 1 than in group 2 and controls. Although LPS was not correlated with study variables, viral load was positively associated with sCD14, LBP, or endotoxin-core antibodies. At the second semester, LPS was not detectable and elevated host LPS-control molecules values were sustained in all groups and in conjunction with ART in all HIV-infected infants.Although plasma concentration of LPS was higher in perinatally HIV-infected infants 0 to 6 months of age than in controls independent of ART initiation strategy, concentration of LPS-control molecules was higher in infants with deferred ART, suggesting the presence of increased microbial translocation in HIV-infected infants with sustained early viral replication.

The incidence of melanoma rises dramatically after the age of 55. Due to an increase in aging population, it is important to study the change in molecular mechanisms due to aging that would allow development of therapies that are tailored to the age of the patients. The role of the tumor microenvironment in modulating cancer characteristics is widely recognized and it also provides targets for therapeutic intervention. Due to this, we hypothesized that changes in tumor microenvironment due to aging could affect the progression of the melanoma. We obtained skin fibroblasts from healthy donors aged 25-35, as well as skin fibroblasts from healthy donors aged 55-65. We cultured these fibroblasts and used conditioned media from them to affect the invasion of melanoma cells in 3D spheroid invasion assays, where aged fibroblasts promoted invasion of melanoma cells into collagen. We also built artificial skin (reconstructs) using young and aged fibroblasts and demonstrated that skin built with aged fibroblasts promoted melanoma cell invasion. Finally using a transgenic mouse model of melanoma (Yumm1.7, BRAFV600E/PTEN-/-) we observed that the injection of melanoma cells into the tail vein of aged mice (52 weeks) formed metastastic colonies much more rapidly than those injected into the tail vein of young mice (8 weeks). To study the factors involved in the aging microenvironment, we performed a proteomics study of the secretome from young and aged fibroblasts. From this study, we observed that aged fibroblasts secreted inhibitors of canonical Wnt signaling, as well as increased deposition of extracellular matrix components in the aging microenvironment. Since inhibition of canonical Wnt signaling has been linked to decreased sensitivity towards BRAF inhibitors in melanoma, we injected Yumm 1.7 cells subcutaneously into aged and young mice. These mice were then treated with BRAF inhibitors. We observed an increased resistance in response to therapy in the aged mice. We also prepared skin reconstruct from fibroblasts with knockdown of the proteins that we identified from secretome and treated them with PLX4720. These results indicated the role of these extracellular matrix proteins in melanoma. We are exploring the mechanisms of how these extracellular matrix proteins affect the sensitivity towards chemotherapeutics. We concluded that aging could alter tumor microenvironment thus resulting in increased metastasis and therapy resistance. It is important that the studies in cancer therapies take into account the age of the patient to achieve better response in patients. Citation Format: Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Michael O'Connell, Marie Webster, Jessica Appleton, Phil Cheng, Alexander Valiga, Rachel Morissette, Nazli McDonnell, Luigi Ferrucci, Andrew Kossenkov, Katrina Meeth, Marcus Bosenberg, Hsin-Yao Tang, Xiangfan Yin, William Wood, III, Elin Lehrmann, Kevin Becker, Keith Flaherty, Dennie Frederick, Jennifer Wargo, Katherine Aird, Rugang Zhang, Xiaowei Xu, Qin Liu, David Speicher, Ashani Weeraratna. Aging microenvironment modulates melanoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A04.

HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4+ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8+ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p

Background The degree of immune reconstitution achieved in response to suppressive ART is associated with baseline individual characteristics, such as pre-treatment CD4 count, levels of viral replication, cellular activation, choice of treatment regimen and gender. However, the combined effect of these variables on long-term CD4 recovery remains elusive, and no single variable predicts treatment response. We sought to determine if adiposity and molecules associated with lipid metabolism may affect the response to ART and the degree of subsequent immune reconstitution, and to assess their ability to predict CD4 recovery. Methods We studied a cohort of 69 (48 females and 21 males) HIV-infected, treatment-naïve South African subjects initiating antiretroviral treatment (d4T, 3Tc and lopinavir/ritonavir). We collected information at baseline and six months after viral suppression, assessing anthropometric parameters, dual energy X-ray absorptiometry and magnetic resonance imaging scans, serum-based clinical laboratory tests and whole blood-based flow cytometry, and determined their role in predicting the increase in CD4 count in response to ART. Results We present evidence that baseline CD4+ T cell count, viral load, CD8+ T cell activation (CD95 expression) and metabolic and anthropometric parameters linked to adiposity (LDL/HDL cholesterol ratio and waist/hip ratio) significantly contribute to variability in the extent of CD4 reconstitution (ΔCD4) after six months of continuous ART. Conclusions Our final model accounts for 44% of the variability in CD4+ T cell recovery in virally suppressed individuals, representing a workable predictive model of immune reconstitution.

Background Advanced HIV infection can result in lipoatrophy and wasting, even in the absence of ongoing opportunistic infections, suggesting that HIV may directly affect adipose tissue amount and distribution. Methods We assessed the relationship of fat (measured using anthropometry, DEXA, MRI scans) or markers related to glucose and lipid metabolism with viral load in a cross-sectional sample of 83 antiretroviral-naïve HIV-1-infected South African women. A multivariable linear model was fitted to log10VL to assess the combined effect of these variables. Results In addition to higher T cell activation, women with viral load greater than the population median had lower waist circumference, body mass index and subcutaneous abdominal fat, as well as lower serum leptin. We demonstrate that leptin serum levels are inversely associated with viral replication, independent of the amount of adipose tissue. This association is maintained after adjusting for multiple variables associated with disease progression (i.e., cellular activation and innate immunity effector levels). Conclusions Our results demonstrate that serum leptin levels are inversely associated with viral replication, independent of disease progression: we postulate that leptin may affect viral replication.