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LD50 of Onc.Ad, HDAd or CAd-VEC in each cell line.

Supplemental Materials and Methods

Supplemental Figure Legends

Anti-PD-L1 antibody and PD-L1 mini-body enhance HER2.CAR T-cell proliferation in vitro.

PD-L1 mini-body is expressed at tumor site 10 days post-injection of HER2.CAR T-cells, but minimally impacts CD4+ T-cell phenotype long-term.

Chimeric antigen receptor–modified T cells (CAR T cells) produce proinflammatory cytokines that increase expression of T-cell checkpoint signals such as PD-L1, which may inhibit their functionality against solid tumors. In this study, we evaluated in human tumor xenograft models the proinflammatory properties of an oncolytic adenovirus (Onc.Ad) with a helper-dependent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody (mini-body; HDPDL1) as a strategy to enhance CAR T-cell killing. Coadministration of these agents (CAd-VECPDL1) exhibited oncolytic effects with production of PD-L1 mini-body locally at the tumor site. On their own, HDPDL1 exhibited no antitumor effect and CAd-VECPDL1 alone reduced tumors only to volumes comparable to Onc.Ad treatment. However, combining CAd-VECPDL1 with HER2.CAR T cells enhanced antitumor activity compared with treatment with either HER2.CAR T cells alone or HER2.CAR T cells plus Onc.Ad. The benefits of locally produced PD-L1 mini-body by CAd-VECPDL1 could not be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T cells and coadministration of Onc.Ad in an HER2+ prostate cancer xenograft model. Overall, our data document the superiority of local production of PD-L1 mini-body by CAd-VECPDL1 combined with administration of tumor-directed CAR T cells to control the growth of solid tumors. Cancer Res; 77(8); 2040–51. ©2017 AACR.

PD-L1 mini-body induces dose-dependent IFNb secretion and T-cell proliferation similar to anti-PD-L1 IgG in MLR.

Systemic treatment of PD-L1 IgG reduces the amount of human T-cells in mice at 24 hrs post-injection.

Co-infection of Onc.Ad with HDPD-L1 increased PD-L1 mini-body in vitro, but PD-L1 mini-body was undetectable in the serum of mice.

Cancer cells express HER2 and PD-L1 expression is induced in the presence of recombinant IFN�, and HER2.CAR T-cells express PD-1 in the presence of target cells.

Psoriasis can greatly impact patients' quality of life. The introduction of new treatments has improved treatment outcomes, but treatment gaps may still exist.Identify unmet treatment needs in patients with psoriasis.Participants aged 18 years or older, with an Amazon Mechanical Turk account, who reported diagnosis of psoriasis and correctly answered an attention check question were included. Results were analyzed using descriptive and inferential statistics.Of the 417 participants who met study inclusion criteria, 51.1% were female, and 39.3% were 31-40 years of age; 61.2% reported mild, 25.4% moderate, and 13.4% severe psoriasis. Most (74.8%) were currently under treatment; half (51.6%) were mostly or completely satisfied with treatment, while 24.5% were slightly or not at all satisfied. Respondents were most satisfied with topical (59.5%), followed by oral (46%), and injectable treatments (19.9%). Most (78.7%) slightly or strongly felt there should be more cost-effective options. Gaps in current psoriasis treatment options included more affordable topical and oral treatments that work faster and require less frequent use.The use of an English survey and Amazon Mechanical Turk precludes certain populations from this study. Participants were not asked to provide their current form of psoriasis treatment.Despite advances in psoriasis treatment, there remains a desire for more effective, faster, longer acting, and less costly, more accessible treatments. J Drugs Dermatol. 2022;21(8):839-844. doi:10.36849/JDD.6589.

For decades, Adenoviruses (Ads) have been staple cancer gene therapy vectors. Ads are highly immunogenic, making them effective adjuvants. These viruses have well characterized genomes, allowing for substantial modifications including capsid chimerism and therapeutic transgene insertion. Multiple generations of Ad vectors have been generated with reduced or enhanced immunogenicity, depending on their intended purpose, and with increased transgene capacity. The latest-generation Ad vector is the Helper-dependent Ad (HDAd), in which all viral coding sequences are removed from the genome, leaving only the cis-acting ITRs and packaging sequences, providing up to 34 kb of transgene capacity. Although HDAds are replication incompetent, their innate immunogenicity remains intact. Therefore, the HDAd is an ideal cancer gene therapy vector as its infection results in anti-viral immune stimulation that can be enhanced or redirected towards the tumor via transgene expression. Co-infection of tumor cells with an oncolytic Ad and an HDAd results in tumor cell lysis and amplification of HDAd-encoded transgene expression. Here, we describe an HDAd-based cancer gene therapy expressing multiple classes of immunomodulatory molecules to simultaneously stimulate multiple axes of immune pathways: the HydrAd. Overall, the HydrAd platform represents a promising cancer immunotherapy agent against complex solid tumors.

Alopecia areata (AA) is a condition characterized by nonscarring hair loss. Cases of alopecia areata are most commonly seen in patients under age 30 and are frequently idiopathic. In this report, we discuss a woman in her 50s who developed AA shortly after receiving the Tdap vaccine and after one year of guselkumab therapy.

There is limited information of the effect of the COVID-19 pandemic on the general population's perceptions towards teledermatology. This study aims to assess the pandemic's impact on people's willingness to use teledermatology as well as to investigate influencing factors. We recruited 544 participants through Amazon Mechanical Turk (MTurk) and surveyed them using REDCap. Participants' willingness to use teledermatology before, during, and after the COVID-19 pandemic was measured via a 5-point Likert scale. The survey also included questions regarding factors influencing participants' attitudes towards teledermatology and their sociodemographic characteristics. Of the 185 participants who reported unwillingness to use teledermatology prior to the COVID-19 pandemic, 79.2% and 66.5% became either neutral or willing to use teledermatology during and after the pandemic, respectively. Less than half of prior satisfactory telemedicine users reported willingness to use teledermatology before the pandemic; willingness to use teledermatology increased to 80.1% and 63.8% during and after the COVID-19 pandemic, respectively. The top reason for lack of interest in teledermatology was concern for security and privacy (24.4%). Although a useful tool, teledermatology has been met with reluctance by the public. However, the unique circumstances of the COVID-19 pandemic have improved the public's perceptions and readiness to use teledermatology.

Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers but has been ineffective for the treatment of microsatellite stable (MSS) PDAC. The lack of efficacy of immunotherapies in PDAC is due to: 1) a desmoplastic tumor microenvironment (TME); 2) the presence of suppressive cells, including myeloid derived suppressor cells and regulatory T cells; and 3) the lack of antigen-presenting dendritic cells (DCs) that are important in priming an effective immune response to generate functionally effective tumor antigen-specific T cells. We recently completed a pilot study of dual ICB (Ipilumamab and Nivolumab) with radiation therapy (SBRT 8Gy for 3 fractions) in a cohort of 25 metastatic PDAC patients that had progressed on conventional chemotherapy; this combination conferred an impressive 18% ORR and 29% disease control rate measured on non-irradiated lesions (historical 0% ORR with ICB in PDAC). This led to a phase 2 study in 30 metastatic PDAC patients using this dual modality treatment paradigm. To understand the role of radiation and ICB in altering the PDAC tumor microenvironment we performed single-cell RNA-sequencing and TCR-sequencing (>180k cells), and single-nucleus RNA-sequencing (>300k cells) on 36 tumor biopsies (23 pre-treatment, 13 paired on-treatment between day 10 and 21) from patients undergoing treatment in our phase 2 study. Tumor tissue was taken from distinct tissue sites, including primary tumors in the pancreas, and liver and abdominal wall metastases. We identified distinct tumor cell state distributions within different tissues, and a redistribution of cells from basal/mesenchymal states to classical states after radiation. We identified several state-specific interferon stimulated gene programs thus cataloging distinct responses of epithelial cells with different transcriptional states. Importantly, we found a redistribution of T cells states towards proliferating and exhausted T cells with unique clonality after radiation. Additionally, the myeloid compartment after radiation was enriched for C1QC+ and MHCII+ macrophage subsets, as well as infiltrating CD16/CD16 monocytes and CD14 monocytes, each showing induction of unique sets of interferon stimulated genes (ISGs). We next sought to better understand immunotherapy resistance mechanisms within these PDAC patients despite finding strong ISG induction in several subsets. We analyzed covarying gene programs and identified multicellular communities of cells before and after radiation that underlie interaction networks associated with radiation. Together our data provides the most comprehensive single-cell atlas of paired biopsies to study tumor and immune cell states in the context of radiation and ICB response. Citation Format: Arnav Mehta, Aparna Parikh, Milan Parikh, Ryan Park, Moshe Sade-Feldman, Lynn Bi, Nicole Carzo, Tarin M. Grillo, Islam Baiev, Olanike Asupoto, Irena Gushterova, Tom LaSalle, Anna Gonye, Emily Blaum, Sebastien Vigneau, Ronan Chaligne, Ana Lako, Thomas Lila, David Nelson, Caroline Porter, Orr Ashenberg, Karthik Jagadesh, William L. Hwang, Christopher Smillie, David P. Ryan, David T. Ting, Theodore Hong, Dana Pe'er, Nir Hacohen. Dissecting the reorganization of pancreatic tumor microenvironments after radiation and immunotherapy reveals insights into immunotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C012.

The goal of the NEC-Zero project is to reduce the burden of necrotizing enterocolitis (NEC) by increasing access to evidence-based tools to help clinicians and parents integrate evidence into daily care. It involves (a) human milk feeding with prioritized mother's own milk; (b) use of a unit-adopted standardized feeding protocol; (c) a unit-adopted strategy for timely recognition that integrates risk awareness and a structured communication tool when symptoms develop; and (d) stewardship of empiric antibiotics and avoidance of antacids. A toolkit for caregivers and parents was developed to make implementation consistent. For clinicians the toolkit includes: the GutCheckNECrisk score, a structured communication tool, the “Avoiding NEC” checklist, and the NEC-Zero website. For parents, NEC-Zero tools include the website, three educational brochures in English and Spanish, and a collaborative care video produced with the NEC Society. This article describes the toolkit and how it has been accessed and used.

Introduction: A small subset of T cells from patients treated with MART-1 engineered adoptive T cell transfer (ACT) were found to co-express Yamanaka transcription factors SOX2, OCT3/4, and NANOG (TSON). Identifying these T cells may have potential use in future T cell based immunotherapy. Methods: Flow (FC) and mass cytometry (MC) were used to evaluate T cells with induced pluripotent stem cell (iPS)-like markers. Peripheral mononuclear cells (PBMC) were collected from 3 melanoma patients (MP) treated with ACT and 1 healthy donor (HD0). Bone marrow (BM) samples were collected from patients with Non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). T cell populations were then screened for stem cell markers: SOX2 OCT3/4 NANOG TRA-1-81 SSEA4 TRA-1-60 CD3+ CD34-. Naïve T cells (CD45RO- CD62L+) from HD0 were exposed to IL-7/IL-15/IL-21 with or without pan-AKT inhibitor (AKTi). These cells were analyzed after 7 days for TSON expression and the following surface markers: CD27 CD28 CD45RO CD45RA CD57 CD62L CD95 CD122 CD127 CCR4 CCR6 CCR7. PBMC collections from 5 healthy donors (HD) were expanded with or without IL-7/IL-15/IL-21 exposure in G-Rex bioreactors for 7 days. TSON expression was analyzed using MC and characterized for the already mentioned markers as well as CXCR3 and Ki67. Lastly, expanded CD95+ cells were single-cell sorted for TSON and their RNA was extracted using the FRISCR method for single-cell RNA sequencing (scRNA-seq). The iPS cell line H1 was included as a positive control. Results: T cells across all 3 MP had a small percentage of TSON expression (0.0002%, 0.0001%, 0.0001%, respectively). This small cohort of T cells co-expressed TRA-1-81, SSEA4, and TRA-1-60 (0.05%, 0.05%, 0.8%, respectively). TSON expression for HD0 was 0.0000013%, while co-expression of TRA-1-81, SSEA4, and TRA-1-60 was undetectable. BM samples NHL and MM also showed TSON expression (0.18% vs 0.06%). Naïve T cells treated with IL-7/IL-15/IL-21 had a significantly higher percentage of TSON after 7 days of exposure compared to T cells treated with IL-7/IL-15/IL-21/AKTi (0.008% vs 0.001%, respectively). Furthermore TSON cell subsets in both treatment populations displayed a phenotype similar to T memory stem cells (TMSC) and central memory T cells (TCM) (CD45RA+ CD27+ CD28+ CD62L+ CCR7+ CD95+ CD57- CD45RO+). Concerning PBMCs from the 5 HD, the proliferation marker Ki67 was displayed on 3 non-stimulated HD and all stimulated HD with a range of median intensities [10, 67]. The presence of CD62L CD27 CD95 CCR7 CD28 CD45RO in both non-stimulated and stimulated cell populations was similar to TCM phenotype. Validation of scRNA-seq is still in process. Early results show that OCT3, NANOG, and cMYC are detected in both TSON and H1 while SOX2 could only be detected in H1. Conclusion: Our data show evidence of T cells with natural TSON expression. However, additional studies are needed to further understand the role of TSON in immunotherapy. Citation Format: Michael Cerniglia, Helena Escuin-Ordinas, Caroline Porter, Maria Alexandrovna Aleshin, Gardenia Cheung-Lau, Zoran Galic, Inbal Abraham-Davidi, Daniel Sanghoon Shin, Orit Rosenblatt-Rosen, Antoni Ribas, Begoña Comin-Anduix. Phenotypic characteristics of T cells co-expressing SOX2, OCT3/4, and NANOG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1551.

Regulatory T cells (Tregs) are an immunosuppressive population that were initially identified as uniformly and stably expressing the transcription factor Foxp3. Tregs have now been further distinguished based on whether they developed in the thymus (thymically-derived Tregs, or tTregs) or in the periphery (peripherally-derived Tregs, or pTregs). Previous studies have shown that deletion of CNS1, a conserved non-coding DNA response element in the Foxp3 locus, leads to selective impairment of pTreg generation without disrupting tTreg generation in the B6 background. However, the role of pTregs versus tTregs in autoimmunity is unclear. Using CRISPR-Cas9, we removed the CNS1 region in the NOD mouse model of spontaneous type 1 diabetes. Based on in vitro Treg generation assays, removal of the CNS1 region impairs Foxp3 induction in naïve NOD CD4+ T cells. However, it did not lead to statistically significant differences in the percent of Tregs found in various tissues, including the spleen, thymus, gut, lymph nodes, and pancreatic islets of non-diabetic mice aged 8–13 weeks. Single-cell RNA seq comparing a 12-week old NOD mouse to a CNS1 KO NOD mouse also did not reveal strong differences in gene expression in the pancreatic islet, gut, or pancreatic lymph node, although flow cytometry analysis does reveal a small but significant decrease in RORyt+ Tregs and an increase in Helios+ Tregs in the gut of CNS1 KO NOD mice. CNS1 KO NOD mice are also being compared to normal NOD mice for type 1 diabetes onset and incidence. These preliminary data suggest that extrathymically-derived Tregs do not play a large role in this mouse model of type 1 diabetes.

Intratumoral treatment with oncolytic adenoviral vectors expressing an immunomodulatory molecule (Armed Onc.Ads) is safe and has shown some clinical benefit in patients with solid tumors. However, local treatment with Armed Onc.Ad has limited anti-tumor effect against metastasized tumors. T cells modified with tumor-directed chimeric antigen receptors (CARs) have shown promise for the systemic treatment of hematological malignancies, but have been less effective in treating solid tumors. Major reasons for this failure include lack of T-cell migration into solid tumors and the inhibitory microenvironments (e.g. PD-L1) at the tumor site. Recent clinical trials with immune-checkpoint inhibitors (e.g. anti-PD-L1 antibody) have broadly enhanced antitumor immunity by improving tumor-specific T cell responses. We therefore hypothesized that an Armed Onc.Ad expressing anti-PD-L1 antibody could enable the blockade of PD-1:PD-L1 interaction between CAR T-cells and cancer cells at the tumor site, and that combining these treatment modalities may have potent and synergistic anti-tumor effect in solid tumors. In this study, we confirmed that PD-L1 is upregulated on squamous cell carcinoma (Pre: 20%, Post: 98%), and prostate cancer cells (Pre: 60%, Post: 100%), in the presence of co-cultured IFNγ producing HER2.CAR T-cells, a population of effectors that we have safely administered to patients with solid tumors. Additionally, HER2.CAR T-cells also express PD-1 upon activation (Pre: 1%, Post: 30%) at 24 hr co-culture. Cancer cell killing by HER2.CAR T-cells was enhanced 2-fold in the presence of anti-PD-L1 IgG in vitro. We constructed a helper-dependent Ad (HDAd: no autonomous replication and no lytic effect) expressing PD-L1 minibody (fused anti-human PD-L1 scFv with human IgG1 constant region (HDPDL1 mini)) and confirmed by western blot that the minibody is secreted in a dose-dependent manner by cancer cells infected with HDPDL1 mini. The avidity of HDAd derived PD-L1 minibody for PD-L1 is similar to commercial anti-PD-L1 IgG, and the reagent enhanced HER2.CAR T-cell killing of target cells by 3-fold compared to HER2.CAR T-cells with control HDAd. Co-infection of Onc.Ad with HDPDL1 mini induces the same oncolytic effect as previously reported, but also induces replication of HDPDL1 mini and thereby markedly increases production of PD-L1 minibody in vitro and in vivo. Intratumoral co-administration of HDPDL1 mini with Onc.Ad induces 4-fold greater expansion of adoptively transferred HER2.CAR T-cells at the tumor site compared to combinatorial treatment of Onc.Ad (without HDPDL1 mini). Our therapy extended median survival from 35 to 70 days compared to HER2.CAR T-cell alone. Our “all-in-one” strategy also appears superior to infusion of anti-PD-L1 IgG, inducing 3-fold greater expansion of HER2.CAR T-cells at the tumor site. Hence, combining HDPDL1 mini with Onc.Ad overcomes a significant immune defense mechanism inhibiting CAR T-cell treatment of solid tumors, and we are now investigating its longer-term benefits.

Background Some individuals remain AIDS-free with a high and stable CD4 cell count without antiretroviral therapy (ART) for many years. We estimated long-term progression-free survival after HIV seroconversion and aimed to identify factors associated with loss of long-term non-progression (LTNP) status. Methods For this cohort study, we used data for individuals with well-estimated dates of HIV-1 seroconversion from the CASCADE Collaboration a network of 28 HIV seroconverter cohort studies in Europe, Australia, Canada, and sub-Saharan Africa. The first cohort began enrolling patients in 1979, and for this analysis we used data pooled in May 1, 2011. We defined non-progression as being HIV-positive without AIDS, ART-naive, and with CD4 counts of 500 cells per mu L or higher. We defined LTNP as non-progression during the first 10 years after seroconversion. We used longitudinal methods to characterise LTNP. Findings Of the 4979 HIV seroconverters in our dataset, 3708 (75%) were men. Median time to progression was 2.07 years (95% CI 1.96-2.17), giving estimated progression-free survivals of 18.4% (17.2-19.6) 5 years, 4.0% (3.6-4.5) 10 years, and 1.4% (0.9-1.5) 15 years after seroconversion. The rate of progression did not change beyond 10 years after seroconversion (0.28 [95% CI 0.26-0.31] per person-year at 10 years after seroconversion, 0.24 [0.19-0.29] per person-year at 15 years, and 0.18 [0.10-0.33] per person-year at 20 years). At 10 years since HIV seroconversion, 283 individuals had LTNP, of whom 202 subsequently lost this status (median time to loss of status 2.49 years [2.05-2.92]). In univariable analyses, loss of LTNP status was associated with CD4 cell count at 10 years after seroconversion (p