Sicca syndrome/Sjögren's disease associated with cancer immunotherapy: a narrative review on clinical presentation, biomarkers, and management.

INTRODUCTION: Almost one-quarter of immune checkpoint inhibitor (ICI) recipients experience sicca syndrome, while Sjögren's disease (SjD) is estimated at 0.3-2.5%, possibly underreported. AREAS COVERED: This narrative review (Medline/Embase until January/31/2024) addresses the pathophysiology, incidence, demographic/clinical features, biomarkers, labial salivary gland biopsy (LSGB), fulfillment of the idiopathic SjD (iSjD) classificatory criteria, differential diagnosis, and management of sicca syndrome/SjD associated with ICIs. EXPERT OPINION: SjD associated with ICIs is underdiagnosed, since studies that performed the mandatory SjD investigation identified that 40-60% of patients with sicca syndrome associated with ICIs meet the iSjD classificatory criteria. LSGB played a fundamental role in recognizing these cases, as most of them had negative anti-Ro/SS-A antibody. Despite the finding of focal lymphocytic sialoadenitis in LSGB samples mimicking iSjD, immunohistochemical analysis provided novel evidence of a distinct pattern for sicca syndrome/SjD associated with ICIs compared to iSjD. The former has scarcity of B lymphocytes, which are a hallmark of iSjD. Additionally, patients with sicca syndrome/SjD associated with ICIs have demographical/clinical/serological and treatment response dissimilarities compared to iSjD. Dryness symptoms are more acute in the former than in iSjD, with predominance of xerostomia over xerophthalmia, and partial/complete response to glucocorticoids. Dryness symptoms in ICI-treated patients warrant prompt SjD investigation.

Late events after anti-CD19 CAR T-cell therapy for relapsed/refractory B-cell non-Hodgkin lymphoma.

Background: The short-term complications from chimeric antigen receptor T-cell therapy (CART) are well characterized, but the long-term complications still need to be further investigated. Therefore, herein, we will review the currently available literature published on the late adverse events following CART. Methods: We reviewed published data available from pivotal trials and real-world experiences with anti-CD19 CART (CART19) for adults with lymphoma. We defined late events as occurring or persisting beyond 1 month after CART infusion. We focused our literature review on the following late-event outcomes post-CART19: cytopenia, immune reconstitution, infections, and subsequent malignancies. Results: Grade 3-4 cytopenia beyond 30 days occurs in 30%-40% of patients and beyond 90 days in 3%-22% of patients and is usually managed with growth-factor and transfusion support, along with neutropenic prophylaxis. B-cell aplasia and hypogammaglobulinemia are expected on-target off-tumor effects of CART19, 44%-53% of patients have IgG < 400 mg/dL, and approximately 27%-38% of patients receive intravenous immunoglobulin (IVIG) replacement. Infections beyond the initial month from CART19 are not frequent and rarely severe, but they are more prevalent and severe when patients receive subsequent therapies post-CART19 for their underlying disease. Late neurotoxicity and neurocognitive impairment are uncommon, and other causes should be considered. T-cell lymphoma (TCL) after CART is an extremely rare event and not necessarily related to CAR transgene. Myeloid neoplasm is not rare post-CART, but unclear causality given heavily pretreated patient population is already at risk for therapy-related myeloid neoplasm. Conclusion: CART19 is associated with clinically significant long-term effects such as prolonged cytopenia, hypogammaglobulinemia, and infections that warrant clinical surveillance, but they are mostly manageable with a low risk of non-relapse mortality. The risk of subsequent malignancies post-CART19 seems low, and the relationship with CART19 and/or prior therapies is unclear; but regardless of the possible causality, this should not impact the current benefit-risk ratio of CART19 for relapsed/refractory B-cell non-Hodgkin lymphoma (NHL).

In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy.

Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted in-silico structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv's structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.

CAR-T cell therapy: a game-changer in cancer treatment and beyond.

In recent years, cancer has become one of the primary causes of mortality, approximately 10 million deaths worldwide each year. The most advanced, chimeric antigen receptor (CAR) T cell immunotherapy has turned out as a promising treatment for cancer. CAR-T cell therapy involves the genetic modification of T cells obtained from the patient's blood, and infusion back to the patients. CAR-T cell immunotherapy has led to a significant improvement in the remission rates of hematological cancers. CAR-T cell therapy presently limited to hematological cancers, there are ongoing efforts to develop additional CAR constructs such as bispecific CAR, tandem CAR, inhibitory CAR, combined antigens, CRISPR gene-editing, and nanoparticle delivery. With these advancements, CAR-T cell therapy holds promise concerning potential to improve upon traditional cancer treatments such as chemotherapy and radiation while reducing associated toxicities. This review covers recent advances and advantages of CAR-T cell immunotherapy.

Incomplete recovery of the CD4+/CD8+ ratio is associated with the late introduction of antiretroviral therapy among people living with HIV infection

ABSTRACT Despite being subject to lower AIDS-related mortality rates and having a higher life expectancy, patients with HIV are more prone to develop non-AIDS events. A low CD4+/CD8+ ratio during antiretroviral therapy identifies people with heightened immune senescence and increased risk of mortality. In clinical practice, finding determinants of a low CD4+/CD8+ ratio may be useful for identifying patients who require close monitoring due to an increased risk of comorbidities and death. We performed a prospective study on the evolution of the CD4+/CD8+ ratio in 60 patients infected with HIV (80% males), who were subjected to two different antiretroviral regimens: early and deferred therapy. The initial CD4+/CD8+ ratio was ≤1 for 70% of the patients in both groups. Older age, CD4+ cell count at inclusion, Nadir CD8+T-cell count, and Initial CD4+/CD8+ ratio ≤ 1 were risk factors for lack of ratio recovery. In the multivariate analysis, a CD4+/CD8+ ratio > 1 at the start of the treatment was found to be a determinant factor in maintaining a CD4+/CD8+ ratio > 1. The nadir CD4+T-cell count was lower in the deferred therapy group (p=0.004), and the last CD4+/CD8+ ratio ≤1 was not associated with comorbidities. Ratio recovery was not associated with the duration of HIV infection, time without therapy, or absence of AIDS incidence. A greater improvement was observed in patients treated early (p=0.003). In contrast, the slope of increase was slower in patients who deferred treatment. In conclusion, the increase in the CD4+/CD8+ ratio occurred mostly for patients undergoing early strategy treatment and its extension did not seem to be related to previous HIV-related factors.

CAR-T Cell Therapy: From the Shop to Cancer Therapy.

Cancer is a worldwide health problem. Nevertheless, new technologies in the immunotherapy field have emerged. Chimeric antigen receptor (CAR) technology is a novel biological form to treat cancer; CAR-T cell genetic engineering has positively revolutionized cancer immunotherapy. In this paper, we review the latest developments in CAR-T in cancer treatment. We present the structure of the different generations and variants of CAR-T cells including TRUCK (T cells redirected for universal cytokine killing. We explain the approaches of the CAR-T cells manufactured ex vivo and in vivo. Moreover, we describe the limitations and areas of opportunity for this immunotherapy and the current challenges of treating hematological and solid cancer using CAR-T technology as well as its constraints and engineering approaches. We summarize other immune cells that have been using CAR technology, such as natural killer (NK), macrophages (M), and dendritic cells (DC). We conclude that CAR-T cells have the potential to treat not only cancer but other chronic diseases.

Autologous, allogeneic hematopoietic cell transplantation and CAR-T/NK therapy: what is their real importance in PTCL?

Peripheral T cell lymphoma (PTCL) is a rare and aggressive type of non-Hodgkin's lymphoma that affects mature T cells. This type of cancer is characterized by the abnormal growth of T cells, which can accumulate in the lymph nodes, spleen, bone marrow, and other organs, leading to a variety of symptoms. PTCLs are often difficult to diagnose and treat, and they have a poorer prognosis than other types of lymphoma. However, recent advancements in treatment options, such as targeted therapies have shown promise in improving outcomes for patients with PTCL. Here, we discuss the use of autologous and allogeneic hematopoietic cell transplantation (HCT) as a treatment strategy for patients with PTCL, as well as the recent treatment approaches based on advanced cellular therapy. The current evidence for the use of HCT in PTCL is mainly derived from registry data, retrospective studies, and expert opinion, as randomized trials are limited due to the low incidence and histological heterogeneity of PTCL subtypes.

Profiles of University Students Who Graduate on Time: A Cohort Study from the Chilean Context.

Graduating from higher education on time is an important topic, given its relation to students' academic success and the efficiency of the institutions. However, a low percentage of university students finish their studies on time, which poses a challenge that requires the identification of the factors that account for this phenomenon. This study aimed to identify and characterize profiles of students who graduate on time. The population is 514 university students (45% men, 55% women), with an average age of 19.5 years (SD= 1.9) studying business at a university in Chile who belong to four cohorts entering between 2011 and 2014. The results obtained from the Classification and Regression Tree (CART) technique demonstrate eight student profiles constructed considering different variables at the pre-university, transition-motivation, and university levels. As the primary outcome, the profile of the student who graduates on time is characterized by a good performance in the first year of university, enters university right after high school, and takes advantage of institutional support by participating in academic tutoring. These findings suggest that institutions can implement specific strategies from the beginning of the university journey to promote on-time graduation.

Prognostic Factors in Children and Adolescents with Lymphomas and Vertical Transmission of HIV in Rio de Janeiro, Brazil: A Multicentric Hospital-Based Survival Analysis Study.

Lymphomas related to HIV are generally aggressive and have a poor prognosis, despite the use of combined antiretroviral therapy (cART) and effective chemotherapy treatment. To determine survival and prognostic factors in children and adolescents living with HIV (CLWH) in Rio de Janeiro (RJ), Brazil, who developed lymphomas, we performed a retrospective and observational study of vertically infected CLWH aged from 0 to 20 incomplete years during1995 to 2018 at five reference centers for cancer and HIV/AIDS treatment. Of the 25 lymphomas, 19 were AIDS-defining malignancies (ADM) and 6 were non-AIDS-defining malignancies (NADM). The 5-year overall survival (OS) and 5-year event-free survival (EFS) probabilities were both 32.00% (95% CI = 13.72-50.23%), and the 5-year disease-free survival (DFS) probability was 53.30% (95% CI = 28.02-78.58%). In the multivariate Cox regression analysis, performance status 4 (PS 4) was considered a poor prognostic factor for OS (HR 4.85, 95% CI = 1.81-12.97, p = 0.002) and EFS (HR 4.95, 95% CI = 1.84-13.34, p = 0.002). For the DFS, higher CD4+ T-cell counts were considered a better prognostic factor (HR 0.86, 95% CI = 0.76-0.97, p = 0.017) in the multivariate Cox regression analysis. This study demonstrates, for the first time, survival and prognostic factors for CLWH who developed lymphomas in RJ, Brazil.

Emerging Targeted Therapies for HER2-Positive Breast Cancer.

Breast cancer is the most common cancer in women and the leading cause of death. HER2 overexpression is found in approximately 20% of breast cancers and is associated with a poor prognosis and a shorter overall survival. Tratuzumab, a monoclonal antibody directed against the HER2 receptor, is the standard of care treatment. However, a third of the patients do not respond to therapy. Given the high rate of resistance, other HER2-targeted strategies have been developed, including monoclonal antibodies such as pertuzumab and margetuximab, trastuzumab-based antibody drug conjugates such as trastuzumab-emtansine (T-DM1) and trastuzumab-deruxtecan (T-DXd), and tyrosine kinase inhibitors like lapatinib and tucatinib, among others. Moreover, T-DXd has proven to be of use in the HER2-low subtype, which suggests that other HER2-targeted therapies could be successful in this recently defined new breast cancer subclassification. When patients progress to multiple strategies, there are several HER2-targeted therapies available; however, treatment options are limited, and the potential combination with other drugs, immune checkpoint inhibitors, CAR-T cells, CAR-NK, CAR-M, and vaccines is an interesting and appealing field that is still in development. In this review, we will discuss the highlights and pitfalls of the different HER2-targeted therapies and potential combinations to overcome metastatic disease and resistance to therapy.

DC vaccine enhances CAR-T cell antitumor activity by overcoming T cell exhaustion and promoting T cell infiltration in solid tumors.

OBJECTIVE: Great success has been achieved in CAR-T cell immunotherapy in the treatment of hematological tumors. However, it is particularly difficult in solid tumors, because CAR-T is difficult to enter interior and exert long-term stable immune effects. Dendritic cells (DCs) can not only present tumor antigens but also promote the infiltration of T cells. Therefore, CAR-T cells with the help of DC vaccines are a reliable approach to treat solid tumors. METHODS: To test whether DC vaccine could promote CAR-T cell therapy in solid tumors, DC vaccine was co-cultured with MSLN CAR-T cells. The in vitro effects of DC vaccine on CAR-T were assessed by measuring cell proliferation, cell differentiation, and cytokine secretion. Effects of DC vaccine on CAR-T were evaluated using mice with subcutaneous tumors in vivo. The infiltration of CAR-T was analyzed using immunofluorescence. The persistence of CAR-T in mouse blood was analyzed using real-time quantitative PCR. RESULTS: The results showed that DC vaccine significantly enhanced the proliferation potential of MSLN CAR-T cells in vitro. DC vaccines not only promoted the infiltration of CAR-T cells, but also significantly improved the persistence of CAR-T in solid tumors in vivo. CONCLUSION: In conclusion, this study has demonstrated that DC vaccine can promote CAR-T therapy in solid tumors, which provides the possibility of widespread clinical application of CAR-T cells in the future.

Chimeric antigen receptor T cells therapy in solid tumors.

Chimeric antigen receptor T cells therapy (CAR-T therapy) is a class of ACT therapy. Chimeric antigen receptor (CAR) is an engineered synthetic receptor of CAR-T, which give T cells the ability to recognize tumor antigens in a human leukocyte antigen-independent (HLA-independent) manner and enables them to recognize more extensive target antigens than natural T cell surface receptor (TCR), resulting in tumor destruction. CAR-T is composed of an extracellular single-chain variable fragment (scFv) of antibody, which serves as the targeting moiety, hinge region, transmembrane spacer, and intracellular signaling domain(s). CAR-T has been developing in many generations, which differ according to costimulatory domains. CAR-T therapy has several limitations that reduce its wide availability in immunotherapy which we can summarize in antigen escape that shows either partial or complete loss of target antigen expression, so multiplexing CAR-T cells are promoted to enhance targeting of tumor profiles. In addition, the large diversity in the tumor microenvironment also plays a major role in limiting this kind of treatment. Therefore, engineered CAR-T cells can evoke immunostimulatory signals that rebalance the tumor microenvironment. Using CAR-T therapy in treating the solid tumor is mainly restricted by the difficulty of CAR-T cells infiltrating the tumor site, so local administration was developed to improve the quality of treatment. The most severe toxicity after CAR-T therapy is on-target/on-tumor toxicity, such as cytokine release syndrome (CRS). Another type of toxicity is on-target/off-tumor toxicity which originates from the binding of CAR-T cells to target antigen that has shared expression on normal cells leading to damage in healthy cells and organs. Toxicity management should become a focus of implementation to permit management beyond specialized centers.

CAR-T cell therapy for multiple myeloma: a practical toolkit for treatment in Brazil.

INTRODUCTION: Chimeric antigen receptor T (CAR-T) cell therapy is an emerging treatment option for relapsed/refractory multiple myeloma (RRMM) that is a multi-step process involving various stakeholders. Appropriate education on the practical logistics is therefore paramount to ensure treatment success. METHODS: A group of key opinion leaders met to explore the key elements of setting up and running a CAR-T center in Brazil. For each step in the CAR-T cell therapy process, the experts agreed on basic requirements, gave their key recommendations from practical experience, and considered any remaining unanswered questions. RESULTS: This paper presents best-practice recommendations and advice on how to overcome common challenges for each step in the CAR-T cell therapy process, with a focus on the current situation in Brazil. Key themes throughout the process are collaboration within the multidisciplinary team and with the referring physician, along with communication and education for patients and their caregivers. CONCLUSION: We believe that the expert insights presented in this paper, in particular on optimal patient selection and timing of CAR-T cell therapy, will deepen understanding of the CAR-T process and aid implementation of this novel therapy for patients with RRMM in Brazil.

P-Glycoprotein and Organic Anion Transporter Polypeptide 1B/Breast Cancer Resistance Protein Drug Transporter Activity in Pregnant Women Living With HIV.

This study evaluates the influence of pregnancy and HIV infection in conjunction with the use of raltegravir, lamivudine, and tenofovir disoproxil fumarate (combined antiretroviral therapy [cART]) on intestinal P-glycoprotein (P-gp) and hepatic organic anion transporter polypeptide (OATP) 1B1/1B3 and/or breast cancer resistance protein (BCRP) drug transporter activity using rosuvastatin (OATP1B/BCRP) and fexofenadine (P-gp) probes. Single oral doses of 5-mg rosuvastatin and 60-mg fexofenadine were administered to women living with HIV under cART in the third trimester of gestation (n = 15) and postpartum period (n = 10). A control group of 12 healthy nonpregnant women also was investigated. Pharmacokinetic parameters were estimated by using a noncompartmental method and evaluated by t test (P < .05). The rosuvastatin area under the plasma concentration-time curve from time 0 to the last quantifiable concentration (AUC0-last ) value was higher in the third trimester of pregnancy (19.5 [95%CI, 16.8-22.3] ng • h/mL] when compared to postpartum (13.3 [95%CI, 9.3-17.5] ng • h/mL), while the fexofenadine AUC0-last values did not differ between the third trimester of pregnancy (738.0 [95%CI, 611.4-864.6] ng • h/mL) and postpartum period (874.9 [95%CI, 408.2-1342.0] ng• h/mL). The rosuvastatin AUC0-last values did not differ between healthy nonpregnant women (13.8 [95%CI, 10.0-17.6] ng • h/mL) and women living with HIV in the postpartum period (13.3 [95%CI, 9.3-17.5] ng • h/mL), and the fexofenadine AUC0-last values did not differ between the 2 investigated groups (603.6 [95%CI, 467.5-739.7] ng • h/mL vs 874.9 [95%CI, 408.2-1342.0] ng • h/mL). It is suggested that gestation inhibits the hepatic OATP1B1/1B3 and/or BCRP activity but does not alter intestinal P-gp activity. The influence of HIV infection in conjunction with use of cART on OATP1B/BCRP and intestinal P-gp activity was not observed.

Defining ambulation status in patients with Duchenne muscular dystrophy using the 10-metre walk test and the motor function measure scale.

PURPOSE: Timed functional tests have been explored to understand the natural history of Duchenne muscular dystrophy (DMD) and to establish warning signs of loss of gait. This study verified whether the combination of the 10-metre walk test (10MWT) and the motor function measure (MFM) could classify the ambulation status of DMD patients. METHOD: Thirty-two patients, aged between 5 and 22 years, with independent gait initially evaluated over 11 years participated in the study. Two groups were created: ambulators and non-ambulators. For both groups, we calculated a 10MWT ratio, by dividing the time spent to perform the last evaluation by the penultimate evaluation, and a MFM dimension-1 score (MFM-D1), collected in the same period. For the statistical analysis, the CART algorithm ("rpart" package in R) classified the patients into ambulators and non-ambulators according to two continuous variables: the 10MWT ratio and the MFM-D1 score. RESULTS: The cut-off points were 1.1 for the 10MWT ratio and 26 points for the MFM-D1, which distinguished 70% of the patients as either ambulators or non-ambulators. CONCLUSION: This simple measurement strategy can be used by therapists to adjust their rehabilitation strategies and goals.Implications for rehabilitationCombination of 10MWT ratio with MFM-D1 reveal an "indicator" for the ambulation status of patients with DMD.Physiotherapists can guide clinical care and prepare the patient and family for loss of gait.CART algorithm describes how we classified the patients according to two continuous variables.70% Of the patients with DMD can be distinguished as either ambulators or non-ambulators.

Childhood leukemias in Mexico: towards implementing CAR-T cell therapy programs.

Leukemias are the most common type of pediatric cancer around the world. Prognosis has improved during the last decades, and many patients are cured with conventional treatment as chemotherapy; however, many patients still present with a refractory disease requiring additional treatments, including hematopoietic stem cell transplantation. Immunotherapy with monoclonal antibodies or cellular therapy is a promising strategy for treating refractory or relapsed hematological malignancies. Particularly, CAR-T cells have shown clinical efficacy in clinical trials, and different products are now commercially approved by regulatory agencies in the USA and Europe. Many challenges still need to be solved to improve and optimize the potential of these therapies worldwide. Global access to cell therapy is a significant concern, and different strategies are being explored in the middle- and low-income countries. In Mexico, leukemias represent around 50% of total cancer diagnosed in pediatric patients, and the rate of relapsed or refractory disease is higher than reported in other countries, a multi-factorial problem. Although significant progress has been made during the last decades in leukemia diagnosis and treatment, making new therapies available to Mexican patients is a priority, and cell and gene therapies are on the horizon. Efforts are ongoing to make CAR-T cell therapy accessible for patients in Mexico. This article summarizes a general landscape of childhood leukemias in Mexico, and we give a perspective about the current strategies, advances, and challenges ahead to make gene and cell therapies for leukemia clinically available.

Impacto do Complexo Repressor Polycomb 2 (PRC2) na modulação in vitro de células T CD8+ humanas / Impact of Polycomb Repressive Complex 2 (PRC2) on the in vitro modulation of human CD8+ T cells

INTRODUÇÃO: Células T CD8+ citotóxicas (CTLs) têm como principal atributo a capacidade de reconhecer e eliminar células-alvo que apresentem epítopos não próprios. Na resposta anti-tumoral, a estimulação persistente ao antígeno pode levar à indução da exaustão de tais células, que passam a apresentar um aumento na expressão de marcadores inibitórios, tornando-as disfuncionais. Nesse contexto, lançamos a hipótese de que mecanismos epigenéticos podem agir como indutores de programas intracelulares envolvidos na supressão de CTLs e que a manipulação farmacológica do epigenoma pode contribuir para a potencialização da resposta citotóxica ao aumentar a capacidade antitumoral dessas células. METODOLOGIA: Foi avaliada inicialmente a atividade modulatória de inibidores de diferentes subunidades do complexo repressor polycomb 2 (PRC2) sobre a função efetora de linfócitos T CD8+ humanos isolados a partir de PBMCs de doadores saudáveis. Além disso, desenvolvemos um sistema de células CAR-T antiCD19 utilizando metodologias não-virais de inserção gênica como o sistema transposontransposase Sleeping Beauty (SB), que representa uma alternativa mais acessível comparada à abordagem tradicional e possibilita a entrega do transgene por eletroporação, agilizando o processo de geração das células CAR-T. RESULTADOS: A inibição das subunidades EED e EZH2 através das sondas epigenéticas A395 e GSK343, respectivamente, aumentou e sustentou ao longo de uma cinética temporal a expressão de marcadores de ativação (CD25 e CD69), mediadores inflamatórios (IFN-γ e TNF-α) e granzima B (GzmB), principal mediador citotóxico produzido por linfócitos T CD8+ , sem que houvesse perda de viabilidade celular. Ademais, a inibição do complexo PRC2 impactou discretamente a proliferação de células T CD8+ , enquanto diminuiu a frequência de marcadores inibitórios como PD1. Utilizando-se dados públicos de expressão gênica de células T CD8+ de camundongos estimuladas in vitro por 72h com anti-CD3/CD28, a deleção condicional de EZH2 promoveu o aumento da expressão gênica de citocinas, quimiocinas e de seus receptores, além da maquinaria de citotoxicidade, o que favoreceu o enriquecimento de vias relacionadas à citotoxicidade celular e à capacidade efetora. Para validarmos o potencial antitumoral citotóxico de linfócitos T CD8+ após a repressão do PRC2, tratamos células CAR-T anti-CD19 com os inibidores GSK343 e A395, os quais também favoreceram o aumento da expressão de mediadores antitumorais e reduziram a expressão de PD1. Além disso, células CAR-T anti-CD19 tratadas com inibidores de PRC2 eliminaram as células de linhagem tumoral de Leucemia Linfoblástica Aguda de células B (LLA-B) CD19+ Nalm-6 mais eficientemente em comparação com células CAR-T anti-CD19 tratadas com os controles negativos. CONCLUSÃO: A inibição do complexo PRC2 potencializou a produção de mediadores inflamatórios e citotóxicos pelas células T CD8 ou células CAR-T. Portanto, nossos resultados sugerem que a modulação epigenética por meio da inibição deste complexo pode ser uma abordagem terapêutica promissora no tratamento oncológico.

INTRODUCTION: Cytotoxic CD8+ T lymphocytes (CTLs) are the main cells responsible for the recognition and elimination of tumor cells. During antitumor responses, chronic antigen stimulation induces CTL exhaustion, which is characterized by the progressive accumulation of inhibitory markers and suboptimal functional properties. Here, we hypothesized that epigenetic mechanisms govern CTL functions, and that pharmacological approaches capable of modulating the CTL epigenome can improve their cytotoxic activity, leading to an improved antitumor response. METHODS: We evaluated the modulatory effect of inhibitors that target different subunits of the Polycomb Repressive Complex 2 (PRC2) on the function of human CD8+ T lymphocytes from healthy PBMCs. In addition, we have developed anti-CD19 CAR-T cells using the Sleeping Beauty (SB) transposon-transposase system, a non-viral gene insertion method. RESULTS: The inhibition of the EED and EZH2 subunits using the epigenetic probes A395 and GSK343 increased and sustained across time the CTL expression of activation markers (i.e., CD25 and CD69), inflammatory mediators (i.e., IFN-γ and TNF-α) and the main cytotoxic granule granzyme B (GzmB), without affecting the CTL viability. In addition, the PRC2 inhibition showed a subtle impact on the CTL proliferation. Furthermore, the use of the epigenetic probes seemed to mitigate the CTL exhaustion process, as seen by the lower expression of PD1 upon PRC2 inhibition when compared with untreated cells. Taking advantage of publicly available transcriptome data from murine CD8+ T cells polyclonally stimulated with anti-CD3/CD28 for 72h, we found that the conditional deletion of EZH2 upregulated the gene expression of cytokines, chemokines/receptors, and the whole cytotoxic machinery, which favored the enrichment of pathways associated with cell cytotoxicity and effector-like cells. In line with this, the PRC2 inhibition of anti-CD19 CAR-T cells also potentiated the expression of antitumor (Gzmb) and inflammatory (IFN-  and TNF-) mediators when compared to the control groups. Moreover, inhibition of the PRC2 complex also seemed to have an impact on CAR-T cell exhaustion, as PD1 was dramatically reduced after the treatment. To validate the antitumor cytotoxic potential of CD8+ T lymphocytes after PRC2 repression, we cocultured CAR-T cells with LLA-B CD19+ Nalm-6 tumor lineage cells and found that the PRC2 inhibition eliminated more efficiently the target cells compared to the control group. CONCLUSION: The PRC2 inhibition potentiated inflammatory and cytotoxic mediators of human lymphocytes and led to improved antitumor activity of CAR-T cells. Therefore, our results suggest that epigenetic modulation through the inhibition of this complex can be a promising therapeutic approach in cancer treatment.

Inativação do receptor PD1 por CRISPR/Cas9 para melhoramento da imunoterapia com células CAR-T

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) é uma técnica de edição genética capaz de editar regiões do DNA de maneira eficaz e relativamente fácil, sendo considerada uma revolução na engenharia genômica. Suas aplicações na área da saúde são diversas. O nocaute pela técnica de CRISPR pode ser aplicado no melhoramento da imunoterapia, em especial de células CAR-T. Considerando os tumores sólidos, estas células são menos eficazes, devido à expressão de moléculas de checkpoint imunológico em células cancerígenas, que ao se ligarem a receptores de superfície em células T, regulam negativamente a resposta imunológica. Um desses receptores é o PD-1, codificado pelo gene PDCD1. Desta forma, este trabalho sugere a inativação do gene PDCD1, visando demonstrar parte do processo necessário para edição e aplicação da técnica CRISPR/Cas9 para o melhoramento da imunoterapia através da inativação do receptor PD-1 em células T.

Factors associated with adherence to antiretroviral therapy in HIV-infected subjects and the use of indicators to characterize the treatment adhesion profile

At present, there is no gold standard to assess patient adherence to combination antiretroviral therapy (cART). Therefore, this study aimed to characterize the epidemiological profile, delineate adherence indicators, and identify factors associated with adherence and delays in obtaining medication in patients registered at the Specialized Assistance Service in HIV/AIDS in Brazil. This is a descriptive study based on secondary data obtained from official databases of the Brazilian Ministry of Health. Adherence and delay were measured by the frequency of cART medication acquisition in 24 months, and a multivariate linear regression model was developed to identify the factors associated with non-adherence and delays. In 50.2% of the subjects, the viral load remained undetectable throughout the study period. Only 12.4% of patients were fully adherent to cART. Regarding indicators, a value of 0.83 was found for adherence, 0.09 for delay in days, and 0.21 for the number of times the patient was late to obtain the medication. The multivariate analysis showed that males, age between 20 and 59 years, having not changed the cART, and the presence of ≥1000 HIV RNA copies/mL were predictive factors for adherence and delays (P≤0.01). We demonstrated that monitoring cART medication distribution is possible using health indicators, and identifying the factors associated with poor adherence to cART helps characterize patients at higher risks of unsuccessful therapy.