Mature tertiary lymphoid structures are key niches of tumour-specific immune responses in pancreatic ductal adenocarcinomas.

OBJECTIVE: To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity. DESIGN: We characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight cancer types. To evaluate the clinical relevance of our findings, we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial. RESULTS: We found that a subset of PDACs harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-ß may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13 + tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens. CONCLUSION: We provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials.

Inibidores epigenéticos como reguladores de linfócitos T CD8+ humanos / Epigenetic inhibitors as regulators of human CD8 T lymphocytes

Linfócitos T CD8 são células chave na resposta antitumoral. Uma vez ativadas, sofrem mudanças epigenéticas, adquirem fenótipos distintos e sua função está ligada ao potencial citotóxico e produção de mediadores inflamatórios. Porém, na resposta antitumoral, tais células encontram-se disfuncionais. Assim, estratégias capazes de reprogramar as células TCD8 podem aumentar sua eficácia. Inibidores epigenéticos são capazes de modular o perfil de células T CD4, mas os efeitos em células T CD8 ainda não estão totalmente esclarecidos. Portanto, buscamos identificar possíveis inibidores epigenéticos que sejam capazes de modular a atividade e o perfil fenotípico de linfócitos T CD8. Para tal, células T CD8 foram isoladas por sorting a partir de PBMC de doadores saudáveis e cultivados em placas de 96 poços na presença de coquetel de ativação (DynaBeads anti-CD3/CD28, IFN- e IL-2 recombinantes) e 1µM de diferentes inibidores epigenéticos (MS023, A485, L-MOSES, A-196, GSKLSD1, A366). Linfócitos T CD8 não estimulados e linfócitos T CD8 ativados na ausência de inibidores epigenéticos foram utilizados como controles. Após 4 ou 8 dias de cultura, as células foram coletadas e analisadas por citometria de fluxo para avaliação de marcadores ligados à ativação (CD69), função (IFN- e granzima B), diferenciação (CCR7, CD45RA) e exaustão (PD-1, TIGIT). As células foram adquiridas no citômetro de fluxo BD FACSymphony A5, e a análise estatística foi feita através do software GraphPad Prism 9. Realizamos ensaios in vitro nos quais células T CD8 foram ativadas na presença ou não de diferentes inibidores, com um deles apresentando intenso potencial de modular células T CD8: o A485, um inibidor seletivo do bromodomínio p300/CBP. Os nossos dados mostram que o A485 aumentou a ativação celular, evidenciado pelo aumento da frequência de células TCD8+CD69+. Este inibidor também foi capaz de modular o fenótipo de memória das células T, polarizando-as para um perfil TN/TSCM, diferentemente do controle e dos outros inibidores, que induziram um perfil TEM (células T efetoras de memória). Após oito dias, o tratamento com o inibidor A485 resultou em menor frequência de células PD-1+ e de células TIGIT+ em comparação com o controle. De relevância clínica, o inibidor A485 foi capaz de modular células CAR-T anti-CD19 isoladas de produtos de infusão, aumentando a frequência do marcador CD69 e modulando seu perfil fenotípico, menos diferenciado, em comparação ao controle. Além disso, o inibidor A485 potencializou a ação antitumoral das células CAR-T anti-CD19 em ensaios de co-cultura com a linhagem celular Daudi CD19+. Em resumo, os nossos dados mostram que a inibição de p300/CBP induz um perfil TN/TSCM em células T CD8 ativadas e em células CAR-T, potencializando as suas atividades antitumorais. Posto que a retenção do perfil TN/TSCM favorece o controle tumoral, esses achados podem ter implicações clínicas para pacientes com doenças hematológicas e com tumores sólidos.

CD8 T lymphocytes are key cells for the induction of antitumor responses. Once activated, these cells undergo epigenetic changes, acquire distinct phenotypes, and their function is linked to the production of cytotoxic and inflammatory mediators. However, these cells are dysfunctional within the tumor microenvironment. Therefore, strategies capable of reprogramming CD8 T cells can enhance their antitumor efficacy. Previous studies have shown that epigenetic inhibitors can modulate the profile of CD4 T cells, but the effects on CD8 T cells are still to be clarified. Thus, we aimed to identify possible epigenetic inhibitors that can modulate the activity and phenotype of CD8 T lymphocytes. To do this, CD8 T lymphocytes were isolated by cell sorting from healthy blood samples and cultured in 96-well plates in the presence of a polyclonal stimulatory cocktail (i.e., anti-CD3/CD28 DynaBeads, recombinant (r)IFN-, and rIL-2) along with 1µM of different epigenetic inhibitors (i.e., MS023, A485, LMOSES, GSKLSD1, and A366). Unstimulated CD8 T lymphocytes and polyclonally-stimulated CD8 T lymphocytes in the absence of epigenetic inhibitors were used as controls. After 4 or 8 days of culture, the cells were analyzed by flow cytometry for the assessment of cell markers related to activation (CD69), function (IFN- and granzyme B), differentiation (CCR7 and CD45RA) and exhaustion (PD-1 and TIGIT). Cells were acquired by the BD FACSymphony A5 cytometer, and statistical analyses were done using the GraphPad Prism 9 software. First, we conducted in vitro assays in which CD8 T cells were activated in the presence or absence of different inhibitors. A485, a p300/CBP bromodomain inhibitor, showed a potent modulatory effect on CD8 T cells. The inhibitor A485 increased T cell activation, observed by the increased frequency of CD69+ CD8 T cells. This inhibitor could also modulate the T cell memory phenotype, polarizing them towards a TN/TSCM profile, unlike the control and the other inhibitors, which polarized them towards to the TEM (effector memory T cells) profile. When the analyses were conducted after eight days of culture, we observed that the treatment with the A485 inhibitor resulted in a lower frequency of PD-1+ and TIGIT+ T cells compared with CD8 T cells activated in the absence of the inhibitor. Of clinical relevance, A485 was able to modulate anti-CD19 CAR-T cells isolated from pre-infusion products by positively regulating the frequency of CD69+ CAR-T cells and modulating their memory phenotype, keeping these cells less differentiated. In addition, A485 potentiated the in vitro antitumor activity of antiCD19 CAR-T cells in co-culture assays with the CD19+ Daudi cell line. In summary, our data show that the p300/CBP inhibition induces the TN/TSCM profile in CD8 T cells and CAR-T cells, thus boosting their antitumor activitiesSince the TN/TSCM phenotype has been shown to be favorable for tumor control, these findings can be of clinical interest for patients with hematological malignancies and solid tumors.

HCV infection prevalence lower than expected among 18-40-year-old injection drug users in San Diego, CA.

San Diego, California shares the world's busiest land border crossing with Tijuana, Mexico-a city where 95 % of injection drug users (IDUs) test hepatitis C virus (HCV) antibody-positive. Yet, little is known about the prevalence and risk behaviors for HCV among IDUs in San Diego. In 2009-2010, 18-40-year-old IDUs in San Diego County completed a risk assessment interview and serologic testing for HCV and HIV infection. Recruitment involved respondent-driven sampling, venue-based sampling at a syringe exchange program, and convenience sampling. Correlates of HCV infection were identified by multivariable logistic regression. Among 510 current IDUs, 26.9 % (95 % CI 23.0-30.7 %) and 4.2 % (95 % CI 2.4-5.9 %) had been infected with HCV and HIV, respectively. Overall, median age was 28 years; 74 % were male; 60 % white and 29 % Hispanic; and 96 % were born in the U.S. Median years of injecting was 6; 41 % injected daily; 60 % injected heroin most often; 49 % receptively shared syringes and 68 % shared other injection paraphernalia; and only 22 % reported always using new syringes in the past 3 months. Two thirds had ever traveled to Mexico and 19 % injected in Mexico. HCV infection was independently associated with sharing injection paraphernalia (adjusted odds ratio [AOR] = 1.69) and SEP use (AOR = 2.17) in the previous 3 months, lifetime history of drug overdose (AOR = 2.66), and increased years of injecting (AOR = 2.82, all P values <0.05). Controlling for recruitment method did not alter results. HCV infection prevalence among IDUs in San Diego was modest compared to other US cities and much lower than Tijuana. Given that known individual-level HCV risk factors were common in San Diego, the city's lower HCV prevalence might be due to differences in social and structural factors between the cities.

Under- and over-nutrition among refugees in San Diego County, California.

Resettled refugees often arrive in their host country with little knowledge of nutrition or available food choices. We explored nutrition-related issues of recent refugee arrivals to San Diego County-the second largest California resettlement site. In-depth interviews (n = 40) were conducted with refugees, health care practitioners, and refugee service organizations. Content analysis identified nutrition-related themes. Unhealthy weight gain after arrival was the most common concern and was attributed to social pressures among adolescents, food choices and a more sedentary lifestyle. Conversely, undernutrition remained a concern due to poor diets. Factors influencing nutritional problems included continuation of past habits, acculturation, unfamiliarity with available foods and socio-economic influences. The nutritional concerns encountered by resettled refugees in San Diego are not unique to this group but are aggravated by their past experiences, and abrupt changes to food choices and behavior. Addressing contextual factors of poor food choices may prevent some of the long term health consequences of poor nutrition.

Latent tuberculosis infection in a migrant agricultural community in Baja California, Mexico.

The objectives were to estimate the prevalence and identify correlates of latent tuberculosis infection (LTBI) among residents of a migrant agricultural community in San Quintín, Baja-California, Mexico. Residents completed a questionnaire and had their blood tested for LTBI using the QuantiFERON(®)-TB Gold In-Tube (QFT) assay. Among 133 participants, 39.8% (95% CI 31.5-48.7%) tested QFT-positive. Having crossed the U.S.-Mexican border since living in San Quintin (P = 0.03), consuming unpasteurized milk (P = 0.02) and receiving health care at IMSS-Oportunidades in the last 6 months (P = 0.03) were independently associated with QFT-positivity. High LTBI prevalence in this community emphasizes the need for TB education and LTBI treatment for its residents. Association with travel to the U.S. suggests the potential for TB transmission across borders. Higher QFT-positivity among those consuming unpasteurized milk could indicate M. bovis infection, previously reported among Mexican migrants living in U.S. border cities.

Young adult injection drug users in the United States continue to practice HIV risk behaviors.

BACKGROUND: Injection drug users (IDUs) are at risk of acquiring HIV through injection and sexual practices. METHODS: We analyzed data collected in five U.S. cities between 2002 and 2004 to identify correlates of HIV infection among 3285 IDUs ages 15-30 years. RESULTS: Overall, HIV prevalence was 2.8% (95% CI 2.3-3.4), ranging from 0.8% in Chicago to 6.3% in Los Angeles. Mean age was 24 years, 70% were male, 64% non-Hispanic (NH) white, 7% NH black, 17% Hispanic, and 12% were other/mixed race. HIV infection was independently associated with: race/ethnicity (NH black [AOR 4.1, 95% CI 1.9-9.1], Hispanic [AOR 3.6, 95% CI 1.5-8.4], or other/mixed [AOR 2.3, 95% CI 1.1-5.2] vs. NH white); males who only had sex with males compared to males who only had sex with females (AOR 15.3, 95% CI 6.8-34.5); injecting methamphetamine alone or with heroin compared to heroin only (AOR 4.0, 95% CI 1.7-9.7); reporting inconsistent means of obtaining income compared to regular jobs (AOR 2.3, 95% CI 1.1-4.8); and having a history of exchanging sex for money/drugs (AOR 2.8, 95% CI 1.5-5.2). CONCLUSIONS: More than two decades after injection and sexual practices were identified as risk factors for HIV infection, these behaviors remain common among young IDUs. While racial/ethnic disparities persist, methamphetamine may be replacing cocaine as the drug most associated with HIV seropositivity. HIV prevention interventions targeting young IDUs and address both sexual and injection practices are needed.