Obesity Influences T CD4 Lymphocytes Subsets Profiles in Children and Adolescent's Immune Response.

BACKGROUND: Evidence shows that CD4+ T cells are altered in obesity and play a significant role in the systemic inflammation in adults with the disease. OBJECTIVES: Because the profile of these cells is poorly understood in the pediatric population, this study aims to investigate the profile of CD4+ T lymphocytes and the plasma levels of cytokines in this population. METHODS: Using flow cytometry, we compared the expression profile of lymphocyte markers, master transcription factors, cytokines, and molecules involved in the regulation of the immune response in CD4+ T cells from children and adolescents with obesity (OB group, n = 20) with those with eutrophy group (EU group, n = 16). Plasma levels of cytokines in both groups were determined by cytometric bead array (CBA). RESULTS: The OB group presents a lower frequency of CD3+ T cells, as well as a decreased frequency of CD4+ T cells expressing CD28, IL-4, and FOXP3, but an increased frequency of CD4+IL-17A+ cells compared with the EU group. The frequency of CD28 is increased in Th2 and Treg cells in the OB group, whereas CTLA-4 is decreased in all subpopulations compared with the EU group. Furthermore, Th2, Th17, and Treg profiles can differentiate the EU and OB groups. IL-10 plasma levels are reduced in the OB group and negatively correlated with adiposity and inflammatory parameters. CONCLUSIONS: CD4+ T cells have an altered pattern of expression in children and adolescents with obesity, contributing to the inflammatory state and clinical characteristics of these patients.

Leukocyte dysfunction and reduced CTLA-4 expression are associated with perianal Crohn's disease.

Although perianal Crohn's disease (PCD) is highly associated with the exacerbated inflammation, the molecular basis and immunological signature that distinguish patients who present a history of perianal lesions are still unclear. This paper aims to define immunological characteristics related to PCD. In this cross-sectional observational study, we enrolled 20 healthy controls and 39 CD patients. Blood samples were obtained for the detection of plasma cytokines and lipopolysaccharides (LPS). Peripheral blood mononuclear cells (PBMCs) were phenotyped by flow cytometry. Leukocytes were stimulated with LPS or anti-CD3/anti-CD28 antibodies. Our results show that CD patients had augmented plasma interleukin (IL)-6 and LPS. However, their PBMC was characterized by decreased IL-6 production, while patients with a history of PCD produced higher IL-6, IL-8, and interferon-γ, along with decreased tumor necrosis factor alpha (TNF). CD patients had augmented FoxP3 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulatory markers, though the PCD subjects presented a significant reduction in CTLA-4 expression. CTLA-4 as well as IL-6 and TNF responses were able to distinguish the PCD patients from those who did not present perianal complications. In conclusion, IL-6, TNF, and CTLA-4 exhibit a distinct expression pattern in CD patients with a history of PCD, regardless of disease activity. These findings clarify some mechanisms involved in the development of the perianal manifestations and may have a great impact on the disease management.

The Expression of CTLA-4 in Breast Tumors and Tumor-Infiltrating Leukocytes Affects Patients' Systemic Inflammatory Status and Varies According to Their Molecular Subtypes.

Recent evidence has pointed out that the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression is a poor prognosis factor. However, the implications of CTLA-4 expression on circulating inflammatory mediators are unclear for breast cancer. Tumor biopsies and blood samples were collected from 117 breast cancer patients. Oxidative stress parameters were evaluated in plasma samples by measuring the lipoperoxidation profile and nitric oxide metabolites (NOx). Interleukins 12 (IL-12) and 4 (IL-4) were assessed by ELISA. CTLA-4 expression was determined by immunofluorescence assessed by its labeling in tumor-infiltrating leukocytes (TILs) or breast tumors. Correlations between CTLA-4 expression in breast tumors with TCD4/TCD8 infiltrating lymphocyte and inflammation-related genes were performed using data from TIMER 2.0/TCGA databases (n = 2160). CTLA-4 expression in TILs significantly correlated to triple-negative breast tumors. Patients carrying CTLA-4-positive tumors exhibited lower plasmatic NOx levels, and those expressing CTLA-4 in TILs had reduced levels of IL-12 in plasma. No changes in either IL-4 or lipid peroxidation profiles were detected concerning any CTLA4 status. Compared to the Luminal A ones, oxidative stress parameters and cytokines were observed in patients bearing triple-negative tumors. CTLA-4 expression in all breast cancer subtypes positively correlated to TCD4/TCD8 lymphocyte infiltrates, as well as to the pro-inflammatory genes IL12A, IL4, NFKB1, NFKB2, NOS1, NOS2, and NOS3. CTLA-4 expression in both tumor and TILs can affect the systemic inflammatory status of breast cancer patients, especially antitumor molecules such as IL-12 and NOx that correlate to more aggressive disease.

Highly purified and functionally stable in vitro expanded allospecific Tr1 cells expressing immunosuppressive graft-homing receptors as new candidates for cell therapy in solid organ transplantation.

The development of new strategies based on the use of Tr1 cells has taken relevance to induce long-term tolerance, especially in the context of allogeneic stem cell transplantation. Although Tr1 cells are currently identified by the co-expression of CD49b and LAG-3 and high production of interleukin 10 (IL-10), recent studies have shown the need for a more exhaustive characterization, including co-inhibitory and chemokines receptors expression, to ensure bona fide Tr1 cells to be used as cell therapy in solid organ transplantation. Moreover, the proinflammatory environment induced by the allograft could affect the suppressive function of Treg cells, therefore stability of Tr1 cells needs to be further investigated. Here, we establish a new protocol that allows long-term in vitro expansion of highly purified expanded allospecific Tr1 (Exp-allo Tr1). Our expanded Tr1 cell population becomes highly enriched in IL-10 producers (> 90%) and maintains high expression of CD49b and LAG-3, as well as the co-inhibitory receptors PD-1, CTLA-4, TIM-3, TIGIT and CD39. Most importantly, high dimensional analysis of Exp-allo Tr1 demonstrated a specific expression profile that distinguishes them from activated conventional T cells (T conv), showing overexpression of IL-10, CD39, CTLA-4 and LAG-3. On the other hand, Exp-allo Tr1 expressed a chemokine receptor profile relevant for allograft homing and tolerance induction including CCR2, CCR4, CCR5 and CXCR3, but lower levels of CCR7. Interestingly, Exp-allo Tr1 efficiently suppressed allospecific but not third-party T cell responses even after being expanded in the presence of proinflammatory cytokines for two extra weeks, supporting their functional stability. In summary, we demonstrate for the first time that highly purified allospecific Tr1 (Allo Tr1) cells can be efficiently expanded maintaining a stable phenotype and suppressive function with homing potential to the allograft, so they may be considered as promising therapeutic tools for solid organ transplantation.

Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-ß in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-ß. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Cytotoxic T-lymphocyte-associated protein 4 in gastric cancer: Prognosis and association with PD-L1 expression.

BACKGROUND: Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the most studied immune checkpoint in gastric cancer (GC). However, the prognostic role of CTLA-4 expression in GC is poorly described. This study aimed to evaluate CTLA-4 expression in GC and its impact on survival, including patients treated with standard platinum-based chemotherapy (CMT), and association with PD-L1 expression. METHODS: All GC patients who underwent D2-gastrectomy were investigated retrospectively. Tumor samples were examined for CTLA-4 and PD-L1 by immunohistochemistry. Tumor-infiltrating inflammatory cells, including CD4 + and CD8 + , were also examined. RESULTS: Among the 284 GC patients included, 159 (56%) were CTLA-4 positive and the remaining 125 (44%) were classified as negative. CTLA-4 positive GC was associated with increased inflammatory cell infiltration (p < 0.001), high CD8 + T cells (p = 0.016) and PD-L1 expression (p = 0.026). Considering GC referred for treatment, CTLA-4 negative patients who received CMT had a significant improvement in disease-free survival compared to untreated CLTA-4 negative (p = 0.028). In multivariate analysis, GC positive for both CTLA-4 and PD-L1 had a prognostic impact on survival. CONCLUSION: CTLA-4 positive was associated with PD-L1 expression and a high tumor-infiltrating CD8 + T cells. Accordingly, positivity for both CTLA-4 and PD-L1 was an independent factor associated to better survival in GC patients.

CTLA-4 Expression and Its Clinical Significance in Breast Cancer.

Breast cancer is the leading cause of women's death among all cancers. The main reason associated with this is the development of metastasis and therapy-resistant breast carcinoma (BC), which pose the main challenge of oncology nowadays. Evidence suggest that these tumors seem to have inhibitory mechanisms that may favor their progression and surveillance. Cancer cells can evade antitumor T cell responses by expressing some immune inhibitory molecules such as the cytotoxic T-lymphocyte antigen-4 (CTLA-4), whose clinical meaning has emerged in the last few years and is poorly understood in the BC context. This systematic literature review aims at identifying studies on CTLA-4 expression in BC, and address what is known about its clinical meaning. A literature search was performed in PubMed and LILACS databases, using the MESH terms "breast cancer"; "CTLA-4 Antigen/antagonists and inhibitors"; and "Lymphocytes, Tumor-Infiltrating/immunology", published in the last 10 years. In total, 12 studies were included in this review. Systematic review used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Despite the small number of eligible studies, the literature reports some associations between CTLA-4 expression in the tumor microenvironment and worse BC outcomes, regardless of its molecular subtype. CTLA-4 expression in BC is a putative marker of clinical significance and a rationale therapeutic target in the emerging field of immunotherapy.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) expression in chordoma and tumor-infiltrating lymphocytes (TILs) predicts prognosis of spinal chordoma.

PURPOSE: Chordoma is a rare tumor of the skeletal system that is characterized by a high recurrence rate and treatment resistance. Given the common finding of immune dysregulation in chordoma, immunotherapy has emerged as potential treatment option. As an important immune checkpoint regulator, we evaluated cytotoxic T-lymphocyte antigen-4 (CTLA-4) expression and its prognostic significance for patients with chordoma of the spine. METHODS: CTLA-4 expression was analyzed immunohistochemically in 32 chordoma tissues and 14 nucleus pulposus tissues to examine the specificity of CTLA-4 expression in chordoma. Univariate log-rank analysis was used to evaluate the association of CTLA-4 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) with survival. Cox multivariate analysis was used to identify independent factors of survival. RESULTS: Positive CTLA-4 expression was observed in all of the TILs and tumor cell cytoplasm, and partial in the membrane or in both the membrane and nucleus, with a markedly higher positivity rate than that observed in normal nucleus tissues. Higher CTLA-4 expression in the tumor but not in TILs was significantly associated with shorter continuous disease-free survival (CDFS) and overall survival (OS). CTLA-4 expression in tumor cells and TILs were independent predictors for CDFS, whereas only tumor cell expression was a significant predictor of OS. Furthermore, the combination of CTLA-4 expression in the tumor and TILs had higher prognostic value. CONCLUSIONS: Targeting CTLA-4 may be a potential novel therapeutic strategy for chordoma patients.

Impact of checkpoint blockade on cancer vaccine-activated CD8+ T cell responses.

Immune and molecular profiling of CD8 T cells of patients receiving DC vaccines expressing three full-length melanoma antigens (MAs) was performed. Antigen expression levels in DCs had no significant impact on T cell or clinical responses. Patients who received checkpoint blockade before DC vaccination had higher baseline MA-specific CD8 T cell responses but no evidence for improved functional responses to the vaccine. Patients who showed the best clinical responses had low PD-1 expression on MA-specific T cells before and after DC vaccination; however, blockade of PD-1 during antigen presentation by DC had minimal functional impact on PD-1high MA-specific T cells. Gene and protein expression analyses in lymphocytes and tumor samples identified critical immunoregulatory pathways, including CTLA-4 and PD-1. High immune checkpoint gene expression networks correlated with inferior clinical outcomes. Soluble serum PD-L2 showed suggestive positive association with improved outcome. These findings show that checkpoint molecular pathways are critical for vaccine outcomes and suggest specific sequencing of vaccine combinations.

In the presence of Trypanosoma cruzi antigens, activated peripheral T lymphocytes retained in the liver induce a proinflammatory phenotypic and functional shift in intrahepatic T lymphocyte.

In secondary lymphoid organs, pathogen-derived and endogenous danger molecules are recognized by pattern recognition receptors, leading to adaptive proinflammatory immune responses. This conceptual rule does not apply directly to the liver, as hepatic immune cells tolerate gut-derived bacterial molecules from the flora. Therefore, the recognition of danger and proinflammatory stimuli differs between the periphery and the liver. However, the tolerant nature of the liver must be overcome in the case of infections or cancer, for example. The central paradigm is the basis for danger recognition and the balance between inflammation and tolerance in the liver. Here, we observed functional integration, with activated peripheral T lymphocytes playing a role in the induction of a proinflammatory environment in the liver in the presence of Trypanosoma cruzi antigens. When only parasite extract was orally administered, it led to the up-regulation of hepatic tolerance markers, but oral treatment plus adoptively transferred activated splenic T lymphocytes led to a proinflammatory response. Moreover, treated/recipient mice showed increased levels of TNF, IFN-γ, IL-6, and CCL2 in the liver and increased numbers of effector and/or effector memory T lymphocytes and F4/80+ cells. There was a reduction in FoxP3+ Treg cells, NKT cells, and γδ T lymphocytes with increased liver damage in the presence of activated peripheral T cells. Our results show that the induction of a proinflammatory liver response against T. cruzi danger molecules is at least partially dependent on cooperation with activated peripheral T cells.

Cervical Cancer Cells Express Markers Associated with Immunosurveillance.

Cervical cancer is the second most frequent cancer in women in Mexico, and its development depends on the presence of human papillomaviruses in the uterine cervix. These oncogenic viruses transform cells where the control over cell cycle disappears, and the capacity to induce apoptosis is absent. On the other hand, some mutations confer to the transformed cells the ability to evade recognition by the immune system. The expression of markers of the immune system such as CD95, MICA/B, CD39, CD73, NKp30, NKp46, CD44, CD24, NKG2A, and CTLA-4 was analysed by flow cytometry on cervical cancer cells INBL (HPV 18, stage IVB), HeLa (HPV 18), CaSki (HPV 16), and C33A (HPV-). Our results showed the presence of atypical markers on cervical cancer cells; some of them are molecules involved in tumour cell recognition such as MICA/B and CD95. Other markers associated with immune system escape, such as CD39, CD73, and CTLA-4, were also present. Furthermore, we found that some cervical cancer cells expressed typical markers of NK cells like NKp30, NKp46, NKG2A, and KIR3DL1. It is not clear whether these molecules confer any gain to the tumour cells or if they represent a disadvantage, but we hypothesise that these molecules that are present in cervical cancer cells allow them to mimic in front of the immune system.

Impaired immunoregulatory network of the CD4 T lymphocytes in refractory asthma.

BACKGROUND: The immunopathogenesis of severe asthma has been associated with an inefficient regulatory response. There are a few studies about the CD4 T cells profile among individuals with severe asthma refractory to treatment. OBJECTIVE: To evaluate the CD4 T lymphocyte profile from individuals with severe asthma according to their response to treatment, relating to their atopy status and age of asthma onset. METHODS: We evaluated nineteen individuals with severe asthma refractory to treatment (SAR), 21 with well-controlled or partly controlled severe asthma (CSA) and 23 with mild-to-moderate asthma (MMA). Lymphocytes were obtained from PBMC, and the frequency of expression of different molecules in this population was assessed using the flow cytometry. RESULTS: We observed the frequency of CD4+ IFN-γ+ T cells was higher in atopic individuals with SAR than with CSA. In addition, among the atopic and early-onset asthma (EOA), the frequency of CD4+ CTLA-4+ T cells was lower in the SAR group than the CSA group. In relation to non-atopic and late-onset asthma (LOA) phenotypes, we noted the frequency of CD4+ FoxP3+ T cells was lower in individuals with SAR than with CSA. We also observed among the LOA patients, the frequency of CD4+ TGF-ß+ T cells was decreased in SAR group than the in CSA group. CONCLUSION AND CLINICAL RELEVANCE: Our data suggest that refractoriness to treatment in asthma is associated with a lower expression of distinct regulatory molecules by CD4 T cells between those who are atopic and have EOA and those who are non-atopic and have LOA. Thus, these results may contribute to the identification of new regulatory strategies to treat asthma according to their phenotypes.

High concentrations of atorvastatin reduce in-vitro function of conventional T and regulatory T cells.

Regulatory T cells (Tregs ) modulate the magnitude of immune responses and possess therapeutic potential in an array of immune diseases. Statins reduce the activation and proliferation of conventional T cells (Tcons ), and they seem to up-regulate the frequency and function of Tregs . However, there is a lack of simultaneous evaluation of the in-vitro effect of statins on the functional profile of Tregs versus Tcons . Herein, magnetically purified Tcons and Tregs were stimulated with CD3/CD28/interleukin (IL)-2 in the presence of atorvastatin (ATV) at 1 or 10 µM. The suppressive function of Tregs , the expression of markers associated with Treg function, activation levels, cytokine production and calcium flux in both subpopulations were assessed by flow cytometry. ATV had no cytotoxic effect on T cells at the concentrations used. Interestingly, 10 µM ATV hampered the suppressive capacity of Tregs . Moreover, this higher concentration reduced the expression of forkhead box protein 3 (FoxP3), cytotoxic T lymphocyte antigen (CTLA-4) and programmed death 1 (PD-1). In Tcons , ATV at 10 µM decreased PD-1 and CD45RO expression. The expression of CD25, CD69, CD95, CD38, CD62L, CCR7 and perforin was not affected in both subpopulations or at any ATV concentrations. Remarkably, 10 µM ATV increased the percentage of tumour necrosis factor (TNF)-α-producing Tregs . Although there was a reduction of calcium flux in Tcons and Tregs , it was only significant in 10 µM ATV-treated Tcons . These results suggested that 10 µM ATV affects the cellular functions of both populations; however, this concentration particularly affected several aspects of Treg biology: its suppressive function, cytokine production and expression of Treg -specific markers.

Immune microenvironment and evasion mechanisms in adenoid cystic carcinomas of salivary glands.

OBJECTIVES: The objective of the present study was to investigate the expression of immune checkpoints (PD-L1, PD-L2, PD-1 and CTLA-4), immune inhibitory molecule HLA-G, markers of tumor-infiltrating lymphocytes (TIL) and dendritic cells (DC), as well as its association with clinicopathological features of adenoid cystic carcinomas (ACC) of the salivary glands. MATERIALS AND METHODS: Thirty-six samples from patients with ACC were analyzed immunohistochemically for the expression of PD-L1, PD-L2, PD-1, CTLA-4, HLA-G, CD8, GrB, CD1a and CD83. Positivity of HLA-G, PD-L1 and PD-L2 expression was defined by cut-offs values. CD8+ TIL was measured semiquantitatively and also using cut-off values obtained by the ROC curve considering recurrence of the lesion. RESULTS: ACC showed low CD8+, GrB+  TIL, CD1a and CD83 populations, as well as scarce positivity for CTLA-4 and PD-1. In contrast, PD-L2 and HLA-G expression was increased, while no PD-L1 expression was detected. Interestingly, cases with lower CD8+ TIL density presented greater recurrence rates. CONCLUSION: Our findings suggest that the ACC microenvironment exhibits low immunogenicity, represented by low TIL and DC density. Moreover, there seems to be activation of the immune inhibitory proteins/PD-L2 and HLA-G, a scenario that may favor tumor escape from the immune system and partially explain the poor prognosis of ACC.

Cancer immunotherapy of patients with HIV infection.

Cancer immunotherapy with antibodies against immune checkpoints has made impressive advances in the last several years. The most relevant drugs target programmed cell death 1 (PD-1) expressed on T cells or its ligand, the programmed cell death ligand 1 (PD-L1), expressed on cancer cells, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Unfortunately, cancer patients with HIV infection are usually excluded from cancer clinical trials, because there are concerns about the safety and the anti-tumoral activity of these novel therapies in patients with HIV infection. Several retrospective studies and some case reports now support the notion that antibodies against immune checkpoints are safe and active in cancer patients with HIV infection, but prospective data in these patients are lacking. In addition, signs of antiviral activity with increase in CD4 T cell counts, plasma viremia reduction or decrease in the viral reservoir have been reported in some of the patients treated, although no patient achieved a complete clearance of the viral reservoir. Here we briefly summarize all clinical cases reported in the literature, as well as ongoing clinical trials testing novel immunotherapy drugs in cancer patients with HIV infection.

CD86 Expression by Monocytes Influences an Immunomodulatory Profile in Asymptomatic Patients with Chronic Chagas Disease.

In the chronic phase of Chagas disease, 60% of the patients develop the asymptomatic form known as indeterminate (IND). The remaining 30% of the patients develop a life-threatening form in which digestive and/or cardiac (CARD) alterations take place. The mechanisms underlying the development of severe forms of Chagas disease remain poorly understood. It is well known that interactions between immune cells such as monocytes and lymphocytes drive immune responses. Further, the co-stimulatory molecules CD80 and CD86 expressed by monocytes and subsets induce lymphocyte activation, thereby triggering cellular immune response. Here, we revealed, for the first time, the functional-phenotypic profile of monocytes subsets in Chagas disease. Using flow cytometry, we evaluated the effect of in vitro stimulation with Trypanosoma cruzi antigens on the expression of the co-stimulatory molecules CD80 and CD86 in different monocyte subsets of patients with IND and CARD clinical forms of Chagas disease. We also assessed the expression of toll-like receptor (TLR)-2, TLR-4, TLR-9, HLA-DR, IL-10, and IL-12 in the monocyte subsets and of CTLA-4 and CD28, ligands of CD80 and CD86, in T lymphocytes. CD86 expression in all monocyte subsets was higher in IND patients when compared with non-infected (NI) individuals. After stimulation with T. cruzi, these patients also showed a higher frequency of CD4+CTLA-4+ T lymphocytes than NI individuals. We found an association between CD80 and CD28, and between CD86 and CTLA-4 expression, with a high frequency of regulatory T (Treg) cells in IND patients. We proposed that CD86 may be involved in immunoregulation by its association with CTLA-4 in asymptomatic patients. CD86 and CTLA-4 interaction may influence Treg activation, and this could represent a new strategy to control inflammation and tissue damage.

Immunotherapy for the treatment of breast cancer.

INTRODUCTION: Breast cancer is the most common cancer as well as the first cause of death by cancer in women worldwide. Although routine treatment improves the outcome of early stage breast cancer patients, there is no effective therapy for the disseminated disease. Immunotherapy has emerged as a powerful therapeutic strategy for the treatment of many cancers. Although traditionally conceived as a non-immunogenic tumor, breast cancer is now considered a potential target for immunotherapy. Areas covered: In this review, the authors discuss different immunotherapeutic strategies that are currently being tested for the treatment of breast cancer: These strategies include: (i) blockade of immunological checkpoints, (ii) antitumor vaccines, (iii) regulatory T cell blockade, (iv) adoptive T cell transfer therapy, (iv) adoptive immunotherapy with monoclonal antibodies, and (v) combination of immunotherapy with chemotherapy. Expert opinion: A growing body of evidence indicates that immunotherapeutic strategies can benefit a larger cohort of breast cancer patients than hitherto anticipated. Since breast tumors entail multiple mechanisms to impair antitumor immunity, the immunological characterization of individual tumors and the selection of suitable combinations of chemotherapeutic and immunotherapeutic approaches are required to achieve significant clinical benefit in these patients.

A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus.

Lymphopenia and autoimmunity: A double-edged sword.

Lymphopenia is strongly associated with autoimmune diseases. The molecular mechanisms that link both phenomena are still unclear, but certain key pathways have been described. Central tolerance is as important as peripheral. In the earlier, epithelial and dendritic cells play a crucial role in the selection of clones. In the latter, regulatory T cells (Tregs) rise as inductors of anergy in order to prevent the development of autoimmune pathology. In lymphopenic conditions, T cells develop the process of lymphopenia-induced proliferation (LIP). A complex interaction between the major histocompatibility complex (MHC) and the T cell receptor (TCR) makes this process possible. Furthermore, IL-7 can act synergistically or in an independent manner to promote LIP. A lack of Transforming Growth Factor-ß (TGF-ß) was recently described as the second hit needed to develop autoimmunity in a lymphopenic microenvironment, given its actions in Tregs and its interaction with CTLA-4. Regarding autoimmune clinical scenarios, lymphopenia is related to both, systemic and organ-specific diseases. Thus, the molecular study of such patients has been limited and needs to be widened to the pathways shown here to be involved in the development of lymphopenia and autoimmunity.