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Follicular CD8 + T cells (fCD8) mediate surveillance in lymph node (LN) germinal centers against lymphotropic infections and cancers, but the precise mechanisms by which these cells mediate immune control remain incompletely resolved. To address this, we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics, and transcriptional profiles of LN-resident virus-specific CD8 + T cells in persons who control HIV without medications. Antigen-induced proliferative and cytolytic potential consistently distinguished spontaneous controllers from noncontrollers. T cell receptor analysis revealed complete clonotypic overlap between peripheral and LN-resident HIV-specific CD8 + T cells. Transcriptional analysis of LN CD8 + T cells revealed gene signatures of inflammatory chemotaxis and antigen-induced effector function. In HIV controllers, the cytotoxic effectors perforin and granzyme B were elevated among virus-specific CXCR5 + fCD8s proximate to foci of HIV RNA within germinal centers. These results provide evidence consistent with cytolytic control of lymphotropic infection supported by inflammatory recruitment, antigen-specific proliferation, and cytotoxicity of fCD8s.

Supplementary Figure 1. Flow chart of patient selection. Supplementary Figure 2. Flowchart of patients with disease response assessment available (n = 57).

This file contains Supplementary Figure 1. Supplementary Figure 1 shows the staining pattern of tumoral PD-L1 staining.

This file contains Supplementary Figure 2. Supplementary Figure 2 shows tumoral PD-L1 staining in association with PD-1, CD8, and CD4 TILs.

This file contains Supplementary Figures 3-9. Supplementary Figure 3. Representative multi-color immunofluorescence staining shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 4. Representative multi-color immunofluorescence staining of a PD-L1 negative tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 5. CD4+ T cells show markedly more specific PD-L1 localization in OTSCC tumors compared to CD8+ T cells. Supplementary Figure 6. Representative multi-color immunofluorescence staining of an OTSCC tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 7. Higher magnification multi-color immunofluorescence staining of a representative OTSCC tumor stained with PD-L1, PD-1, CD4, and CD8. Supplementary Figure 8. Diffuse tumoral PD-L1 staining shows low infiltration of CD4+ and CD8+ T cells. Supplementary Figure 9. Representative multi-color immunofluorescence staining of FoxP3, PD1, and Ki67 in OTSCC.

Purpose:Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation.Experimental Design:This study examined circulating BRAF, NRAS, and c-KIT mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor–based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma).Results:Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume (P < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response (P < 0.01) but not intracranial response. The median overall survival in patients with undetectable (n = 34) versus detectable (n = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28–0.94; P = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16–0.63; P < 0.01).Conclusions:ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma.

This file contains Supplementary Table 1. Supplementary Table 1 shows the immunohistochemical staining for PD-L1, PD-1, CD4, and CD8 in the samples analyzed in the manuscript.

This file contains Supplementary Table 2. Supplementary Table 2 shows that the proliferative capacity of PD-1 expressing CD8, CD4, and FoxP3+ TILs are decreased compared to non-PD-1 expressing TILs in OTSCC.

This file contains the figure legends for supplementary figures 1-9. Supplementary Figure 1. Staining pattern of tumoral PD-L1 staining. Supplementary Figure 2. Tumoral PD-L1 staining in association with PD-1, CD8, and CD4 TILs. Supplementary Figure 3. Representative multi-color immunofluorescence staining shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 4. Representative multi-color immunofluorescence staining of a PD-L1 negative tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 5. CD4+ T cells show markedly more specific PD-L1 localization in OTSCC tumors compared to CD8+ T cells. Supplementary Figure 6. Representative multi-color immunofluorescence staining of an OTSCC tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 7. Higher magnification multi-color immunofluorescence staining of a representative OTSCC tumor stained with PD-L1, PD-1, CD4, and CD8. Supplementary Figure 8. Diffuse tumoral PD-L1 staining shows low infiltration of CD4+ and CD8+ T cells. Supplementary Figure 9. Representative multi-color immunofluorescence staining of FoxP3, PD1, and Ki67 in OTSCC.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity tumor. In this study, we examined the basis for the activity of programmed cell death protein (PD-1)-based immune checkpoint therapy that is being explored widely in head and neck cancers. Using multispectral imaging, we systematically investigated the OTSCC tumor microenvironment (TME) by evaluating the frequency of PD-1 expression in CD8+, CD4+, and FoxP3+ tumor-infiltrating lymphocytes (TIL). We also defined the cellular sources of PD-1 ligand (PD-L1) to evaluate the utility of PD-1:PD-L1 blocking antibody therapy in this patient population. PD-L1 was expressed in 79% of the OTSCC specimens examined within the TME. Expression of PD-L1 was associated with moderate to high levels of CD4+ and CD8+ TILs. We found that CD4+ TILs were present in equal or greater frequencies than CD8+ TILs in 94% of OTSCC and that CD4+FOXP3neg TILs were colocalized with PD-1/PD-L1/CD68 more frequently than CD8+ TILs. Both CD4+PD1+ and CD8+PD1+ TILs were anergic in the setting of PD-L1 expression. Overall, our results highlight the importance of CD4+ TILs as pivotal regulators of PD-L1 levels and in determining the responsiveness of OTSCC to PD1-based immune checkpoint therapy. Cancer Res; 77(22); 6365–74. ©2017 AACR.

This file contains Supplementary Table 3. Supplementary Table 3 shows the clinicopathological characteristics of the patient cohort.

Neuroimaging studies point to neurostructural abnormalities in youth with anxiety disorders. Yet, findings are based on small-scale studies, often with small effect sizes, and have limited generalizability and clinical relevance. These issues have prompted a paradigm shift in the field towards highly powered (i.e., big data) individual-level inferences, which are data-driven, transdiagnostic, and neurobiologically informed. Here, we built and validated neurostructural machine learning (ML) models for individual-level inferences based on the largest-ever multi-site neuroimaging sample of youth with anxiety disorders (age: 10-25 years, N=3,343 individuals from 32 global sites), as compiled by three ENIGMA Anxiety Working Groups: Panic Disorder (PD), Generalized Anxiety Disorder (GAD), and Social Anxiety Disorder (SAD). ML classifiers were trained on MRI-derived regional measures of cortical thickness, surface area, and subcortical volumes to classify patients and healthy controls (HC) for each anxiety disorder separately and across disorders (transdiagnostic classification). Modest, yet robust, classification performance was achieved for PD vs. HC (AUC=0.62), but other disorder-specific and transdiagnostic classifications were not significantly different from chance. However, above chance-level transdiagnostic classifications were obtained in exploratory subgroup analyses of male patients vs. male HC, unmedicated patients vs. HC, and patients with low anxiety severity vs. HC (AUC 0.59-0.63). The above chance-level classifications were based on plausible and specific neuroanatomical features in fronto-striato-limbic and temporo-parietal regions. This study provides a realistic estimate of classification performance in a large, ecologically valid, multi-site sample of youth with anxiety disorders, and may as such serve as a benchmark.

Viruses are the second leading cause of cancer worldwide, and human papillomavirus (HPV)-associated head and neck cancers are increasing in incidence in the United States. HPV preferentially infects the crypts of the tonsils rather than the surface epithelium. The present study sought to characterize the unique microenvironment within the crypts to better understand the viral tropism of HPV to a lymphoid-rich organ. Laser-capture microdissection of distinct anatomic areas (crypts, surface epithelium, and germinal centers) of the tonsil, coupled with transcriptional analysis and multiparameter immunofluorescence staining demonstrated that the tonsillar crypts are enriched with myeloid populations that co-express multiple canonical and noncanonical immune checkpoints, including PD-L1, CTLA-4, HAVCR2 (TIM-3), ADORA2A, IDO1, BTLA, LGALS3, CDH1, CEACAM1, PVR, and C10orf54 (VISTA). The resident monocytes may foster a permissive microenvironment that facilitates HPV infection and persistence. Furthermore, the myeloid populations within HPV-associated tonsil cancers co-express the same immune checkpoints, providing insight into potential novel immunotherapeutic targets for HPV-associated head and neck cancers.

The clinical significance of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA in stool remains uncertain. We found that extrapulmonary dissemination of infection to the gastrointestinal tract, assessed by the presence of SARS-CoV-2 RNA in stool, is associated with decreased coronavirus disease 2019 (COVID-19) survival. Measurement of SARS-CoV-2 RNA in stool may have utility for clinical risk assessment.

Suicide risk is elevated in psychiatric patients following discharge from inpatient care. Despite this vulnerability, there has been limited research investigating suicide prevention protocols that take into account the unique system characteristics of this setting (e.g., short lengths of stay, crisis stabilization treatment model, multidisciplinary team coordination). Cognitive-behavioral therapy (CBT) has demonstrated efficacy for improving suicide risk in outpatients, but has not been validated with inpatients. The current study was a treatment development and pilot feasibility open trial that modified brief cognitive-behavioral therapy (BCBT) for an inpatient setting (BCBT-I). Key treatment modifications included administering up to 10 sessions (depending on patient length of stay), daily, and in a standardized order, with core crisis management skills introduced during the first three sessions. In addition, coordination with the inpatient treatment team was included in BCBT-I implementation. Six adult inpatients with a recent suicide attempt enrolled and completed an average of 4.67 BCBT-I sessions (SD = 1.36). The treatment was highly acceptable (Client Satisfaction Questionnaire total score M = 3.49, SD = 0.73). Pre- to posttreatment effect sizes demonstrated improvements in suicidal ideation (d = 0.97), depression (d = 1.33), and suicidal implicit associations (d = 1.28). All but one of the participants (83%) completed follow-up assessments 1-, 2-, and 3-months postdischarge. Over follow-up, two participants reported suicidal ideation (both without intent), and none reported suicide attempts, preparatory acts or behaviors, or nonsuicidal self-injury. This study provided preliminary evidence supporting the feasibility of CBT to treat suicidal inpatients. Future research is needed to validate BCBT-I in a larger, randomized controlled trial to determine whether BCBT-I reduces suicide risk beyond that afforded by inpatient treatment alone.

Cognitive reappraisal is associated with reduced emotional distress; however, little is known about the nature of this relationship in autism. This study tested whether autistic traits moderate reappraisal success (i.e., the negative correlation between reappraisal use and emotional symptom severity). Emotional symptoms were assessed using measures of depression, anxiety, and stress. It was hypothesized that more severe autistic traits would be associated with weaker reappraisal success across all scales. Data were collected from 377 adults using an on-line survey. Structural equation models found moderation effects for depression and anxiety, but not stress. Contrary to hypotheses, more severe autistic traits were associated with stronger reappraisal success. These preliminary results support including reappraisal in emotion regulation treatments for individuals with autistic traits.

Alveolar macrophages (AMs) are critical for defense against airborne pathogens and AM dysfunction is thought to contribute to the increased burden of pulmonary infections observed in individuals living with HIV-1 (HIV). While HIV nucleic acids have been detected in AMs early in infection, circulating HIV during acute and chronic infection is usually CCR5 T cell-tropic (T-tropic) and enters macrophages inefficiently in vitro. The mechanism by which T-tropic viruses infect AMs remains unknown. We collected AMs by bronchoscopy performed in HIV-infected, antiretroviral therapy (ART)-naive and uninfected subjects. We found that viral constructs made with primary HIV envelope sequences isolated from both AMs and plasma were T-tropic and inefficiently infected macrophages. However, these isolates productively infected macrophages when co-cultured with HIV-infected CD4+ T cells. In addition, we provide evidence that T-tropic HIV is transmitted from infected CD4+ T cells to the AM cytosol. We conclude that AM-derived HIV isolates are T-tropic and can enter macrophages through contact with an infected CD4+ T cell, which results in productive infection of AMs. CD4+ T cell-dependent entry of HIV into AMs helps explain the presence of HIV in AMs despite inefficient cell-free infection, and may contribute to AM dysfunction in people living with HIV.

Sjögren's syndrome is a chronic autoimmune disorder whose pathogenesis is poorly understood and that lacks effective therapies. Detailed quantitative and spatial analyses of tissues affected by Sjögren's syndrome were undertaken, including the quantitation of the frequency of selected cell-cell interactions in the disease milieu. Quantitative analyses of CD4

Individuals with generalized anxiety disorder (GAD) report poorer quality of life (QOL) than do nonanxious controls. Further, although positive affect (PA) and negative affect (NA) have been shown to predict QOL, no previous literature has tested this relationship in the context of individuals with GAD. In the present study, we evaluated the unique and interactive contributions of PA and NA on QOL within a sample of individuals diagnosed with GAD (N = 50). Specifically, a hierarchical regression was conducted to evaluate the unique contributions of PA, NA, and their interaction on QOL, over and above symptoms of depression. PA and depression symptoms were both significant predictors of QOL, whereas neither the main effect for NA nor the PA × NA interaction was statistically significant. Results suggest that, for those with GAD, PA uniquely contributes to QOL. Strategies to upregulate PA may be a useful treatment target for increasing QOL in individuals with GAD.

Objectives Assessing late-life anxiety using an instrument with sound psychometric properties including cross-cultural invariance is essential for cross-national aging research and clinical assessment. To date, no cross-national research studies have examined the psychometric properties of the frequently used Geriatric Anxiety Inventory (GAI) in depth. Method Using data from 3,731 older adults from 10 national samples (Australia, Brazil, Canada, The Netherlands, Norway, Portugal, Spain, Singapore, Thailand, and United States), this study used bifactor modeling to analyze the dimensionality of the GAI. We evaluated the “fitness” of individual items based on the explained common variance for each item across all nations. In addition, a multigroup confirmatory factor analysis was applied, testing for measurement invariance across the samples. Results Across samples, the presence of a strong G factor provides support that a general factor is of primary importance, rather than subfactors. That is, the data support a primarily unidimensional representation of the GAI, still acknowledging the presence of multidimensional factors. A GAI score in one of the countries would be directly comparable to a GAI score in any of the other countries tested, perhaps with the exception of Singapore. Discussion Although several items demonstrated relatively weak common variance with the general factor, the unidimensional structure remained strong even with these items retained. Thus, it is recommended that the GAI be administered using all items.

Hoarding disorder (HD) is a new psychiatric diagnosis in Diagnostic and Statistical Manual of Mental Disorders–Fifth edition and preliminary evidence suggests that cognitive-behavioral treatments are effective in treating this condition. However, it has been demonstrated that individuals with HD generally display poor compliance during treatment, which may lead to poor outcomes. Treatment compliance can be conceptualized as either within-session or between-session compliance, but currently there are no validated measures of within-session or between-session compliance specifically for HD. The aim of this study was to provide an initial validation of the CBT Compliance Measure and the Patient Exposure/Response Prevention Adherence Scale for Hoarding in a sample of participants with HD who were undergoing group cognitive behavioral therapy (CBT) for HD ( N = 70). Both measures, which were administered at each relevant treatment session, demonstrated a unidimensional structure, good reliability, as well as predictive validity, and are thus promising in the measurement of within-session and between-session compliance with CBT for HD.

Circulating cancer-derived small extracellular vesicles (EVs) are nanoscale membranous vesicles shed from cancer cells that are released into surrounding body fluids. Small EVs contain biomolecules associated with cancer such as DNA and proteins for cell-to-cell communication. Therefore, small EVs have been regarded as important cancer biomarkers for liquid biopsy-based cancer diagnosis and drug treatment monitoring. However, because of the high heterogeneity and low level of small EVs in body fluids, there is a high demand for sensitive detection and characterization of such vesicles at a molecular level. In this study, we have developed a sensitive and effective approach to simultaneously profile multiple protein biomarkers expressed on cancer-derived small EVs using surface-enhanced Raman spectroscopy (SERS) nanotags in a single test, without complex isolation steps. Rapid and multiplexed phenotypic profiling of small EVs is achieved by mixing specific detection antibody-coated SERS nanotags, filtered conditioned EV-suspended medium (conditioned EVs), and capture antibody (CD63)-conjugated magnetic beads to form a sandwich immunoassay. As a proof-of-concept demonstration, we applied this approach to characterize pancreatic cancer-derived EVs by simultaneously detecting three specific EV surface receptors including Glypican-1, epithelial cell adhesion molecules (EpCAMs), and CD44 variant isoform 6 (CD44V6). The sensitivity of this method was measured down to 2.3 × 106 particles/mL, which is more sensitive and shows higher multiplexing capability than most other reported EV profiling techniques, such as western blot, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, phenotypic profiling of small EVs from colorectal cancer and bladder cancer cell lines (SW480 and C3) was conducted and compared to those derived from pancreatic cancer (Panc-1), highlighting the significant difference in EV phenotypes for various cancer cell types suspended in both phosphate-buffered saline and plasma. Thus, we believe that this technology enables a comprehensive evaluation of small secreted EV heterogeneity with high sensitivity, offering strong potential for accurate noninvasive cancer diagnosis and monitoring of drug treatment. In addition, this assay provides point-of-care use because of the easy sample preparation and portable nature of the Raman spectrometer.

Blue nevi are benign, melanocytic neoplasms that show a range of clinical and morphologic patterns and include common/dendritic, cellular, and atypical cellular subtypes. Like other nevi, they most commonly occur in skin but can occasionally involve lymph nodes where they may be misinterpreted as representing metastatic melanoma. Moreover, whether benign blue nevi can metastasize to lymph nodes and their natural history and prognostic significance has been the subject of great controversy. To date, few cases of nodal blue nevi have been reported in the literature, and those reports have had limited clinical follow-up and supporting molecular data. This study sought to determine the clinical, pathologic, and molecular features of blue nevi involving lymph nodes, clarify their clinical significance, provide evidence for understanding their pathogenesis, and highlight potential pitfalls in the interpretation of lymph nodes with an ultimate aim of improving patient care. Thirteen cases of blue nevi involving lymph nodes were identified in the archives of Royal Prince Alfred Hospital, Sydney, Australia (1984-2018). A detailed assessment of the clinical and pathologic features of each case was performed, including an evaluation of all available immunohistochemical stains. Extended clinical follow-up was available for 9 patients. Droplet digital polymerase chain reaction for GNAQ Q209L, Q209P and GNA11 Q209L mutations was performed on 7 cases of blue nevi within lymph nodes together with matching cutaneous (presumed primary) blue nevi in 2 cases. All cases showed typical histologic features of blue nevi. BAP1 was retained in all cases (n=7). There were no recurrence or metastasis of blue nevus in any case on long-term clinical follow-up (n=9, median follow-up, 12 y). The majority of cases (n=5 of 7 evaluated) had GNAQ and GNA11 driver mutations. The 2 patients with a matched primary cutaneous blue nevus and regionally associated nodal blue nevus had the same GNAQ Q209L mutation in both sites in each patient. We conclude that blue nevi can involve lymph nodes and are associated with benign clinical behavior, and probably represent so-called "benign" metastasis. Awareness of these lesions is important when evaluating lymph nodes to avoid misdiagnosis as metastatic melanoma.

The aim of the present study was to examine self-report, peripheral nervous system, and central nervous system correlates of naturally-occurring, chronic hyperventilation (HV, assessed by hypocapnia or low resting state low end-tidal CO