Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8+ T cells.

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.

Characterization of Ly108-H1 Signaling Reveals Ly108-3 Expression and Additional Strain-Specific Differences in Lupus Prone Mice.

Ly108 (SLAMF6) is a homophilic cell surface molecule that binds SLAM-associated protein (SAP), an intracellular adapter protein that modulates humoral immune responses. Furthermore, Ly108 is crucial for the development of natural killer T (NKT) cells and CTL cytotoxicity. Significant attention has been paid towards expression and function of Ly108 since multiple isoforms were identified, i.e., Ly108-1, Ly108-2, Ly108-3, and Ly108-H1, some of which are differentially expressed in several mouse strains. Surprisingly, Ly108-H1 appeared to protect against disease in a congenic mouse model of Lupus. Here, we use cell lines to further define Ly108-H1 function in comparison with other isoforms. We show that Ly108-H1 inhibits IL-2 production while having little effect upon cell death. With a refined method, we could detect phosphorylation of Ly108-H1 and show that SAP binding is retained. We propose that Ly108-H1 may regulate signaling at two levels by retaining the capability to bind its extracellular as well as intracellular ligands, possibly inhibiting downstream pathways. In addition, we detected Ly108-3 in primary cells and show that this isoform is also differentially expressed between mouse strains. The presence of additional binding motifs and a non-synonymous SNP in Ly108-3 further extends the diversity between murine strains. This work highlights the importance of isoform awareness, as inherent homology can present a challenge when interpreting mRNA and protein expression data, especially as alternatively splicing potentially affects function.

Discrimination of foreign bodies in quinoa (Chenopodium quinoa Willd.) grains using convolutional neural networks with a transfer learning approach.

The rising interest in quinoa (Chenopodium quinoa Willd.) is due to its high protein content and gluten-free condition; nonetheless, the presence of foreign bodies in quinoa processing facilities is an issue that must be addressed. As a result, convolutional neural networks have been adopted, mostly because of their data extraction capabilities, which had not been utilized before for this purpose. Consequently, the main objective of this work is to evaluate convolutional neural networks with a learning transfer for foreign bodies identification in quinoa samples. For experimentation, quinoa samples were collected and manually split into 17 classes: quinoa grains and 16 foreign bodies. Then, one thousand images were obtained from each class in RGB space and transformed into four different color spaces (L*a*b*, HSV, YCbCr, and Gray). Three convolutional neural networks (AlexNet, MobileNetv2, and DenseNet-201) were trained using the five color spaces, and the evaluation results were expressed in terms of accuracy and F-score. All the CNN approaches compared showed an F-score ranging from 98% to 99%; both color space and CNN structure were found to have significant effects on the F-score. Also, DenseNet-201 was the most robust architecture and, at the same time, the most time-consuming. These results evidence the capacity of CNN architectures to be used for the discrimination of foreign bodies in quinoa processing facilities.

Amazonian cacao-clone nibs discrimination using NIR spectroscopy coupled to naïve Bayes classifier and a new waveband selection approach.

Near-Infrared Spectroscopy (NIRS) has shown to be helpful in the study of rice, tea, cocoa, and other foods due to its versatility and reduced sample treatment. However, the high complexity of the data produced by NIR sensors makes necessary pre-treatments such as feature selection techniques that produce compact profiles. Supervised and unsupervised techniques have been tested, creating different subsets of features for classification, which affect the performance of the classifiers based on such compact profiles. In this sense, we propose and test a new covering array feature selection (CAFS) algorithm coupled to the naïve Bayes classifier (NBC) to discriminate among Amazonian cacao nibs from six cacao clones. The CAFS wrapper approach looks for the wavebands that maximize the F1-score, and then, are more relevant for classification. For this purpose, cacao pods of six varieties were collected, and their grains were extracted and processed (fermented, dried, roasted, and milled) to obtain cacao nibs. Then from each clone NIR spectral profiles in the range of 1100-2500 nm were extracted, and relevant wavebands were selected using the proposed CAFS algorithm. For comparison, two standard feature selection techniques were implemented the multi-cluster feature selection MCFS and the eigenvector centrality feature selection ECFS. Then, based on the different selected variables, three NBCs were built and compared among them through statistical metrics. The results showed that using the wavebands selected by CAFS, the NBC performed an average accuracy of 99.63%; being this superior to the 94.92% and 95.79% for ECFS and MCFS respectively. These results showed that the wavebands selected by the proposed CAFS algorithm allowed obtaining a better fit concerning other feature selection methods reported in the literature.

Myeloid antigen-presenting cell niches sustain antitumor T cells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

Characterization of whey protein-based films incorporated with natamycin and nanoemulsion of α-tocopherol.

Food packaging materials are commonly derived from petroleum that increases global contamination; this raises the interest to evaluate raw material from renewable sources such as whey protein for the development of packaging materials, especially to produce active films. This research aimed to evaluate whey protein-based film properties when natamycin, nanoemulsioned α-tocopherol, or both were added. An oil-in-water (O/W) nanoemulsion of antioxidant (α-tocopherol) was prepared by microfluidization technique. Four films were prepared with different levels of natamycin and nanoemulsified α-tocopherol and were characterized in terms of physicochemical, mechanical, optical-properties, water vapor barrier, FTIR, microstructure, antioxidant and antimicrobial activity. The natamycin, nanoemulsified α-tocopherol, or both did not modify the moisture content of the films. Moreover lead to a significant reduction of tensile strength and elastic modulus, while presenting growth in the elongation at break. Film opacity, the total color difference, the UV-Vis light barrier, and the water vapor permeability values increased when compounds were incorporated into the film. The microstructure studies showed uniformly distributed porosity throughout the films. The addition of nanoemulsioned α-tocopherol into whey protein-based films provoked antioxidant activity and the addition of natamycin produced films with effectivity against C. albicans, P. chrysogenum, and S. cerevisiae, allowing develop a material appropriate for use as active food packaging.

Determination of the superficial citral content on microparticles: An application of NIR spectroscopy coupled with chemometric tools.

This work evaluates near-infrared (NIR) spectroscopy coupled with chemometric tools for determining the superficial content of citral ( S C C t ) on microparticles. To perform this evaluation, using spray drying, citral was encapsulated in a matrix of dextrin using twelve combinations of citral:dextrin ratios (CDR) and inlet air temperatures (IAT). From each treatment, six samples were extracted, and their S C C t and NIR absorption spectral profiles were measured. Then, the spectral profiles, pretreated and randomly divided into modeling and validation datasets, were used to build the following prediction models: principal component analysis-multilinear regression (PCA-MLR), principal component analysis-artificial neural network (PCA-ANN), partial least squares regression (PLSR) and an artificial neural network (ANN). During the validation stage, the models showed R 2 values from 0.73 to 0.96 and a root mean squared error (RMSE) range of [0.061-0.140]. Moreover, when the models were compared, the full and optimized ANN models showed the best fits. According to this study, NIR coupled with chemometric tools has the potential for application in determining S C C t on microparticles, particularly when using ANN models.

The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity.

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence.

Multilayer perceptron architecture optimization using parallel computing techniques.

The objective of this research was to develop a methodology for optimizing multilayer-perceptron-type neural networks by evaluating the effects of three neural architecture parameters, namely, number of hidden layers (HL), neurons per hidden layer (NHL), and activation function type (AF), on the sum of squares error (SSE). The data for the study were obtained from quality parameters (physicochemical and microbiological) of milk samples. Architectures or combinations were organized in groups (G1, G2, and G3) generated upon interspersing one, two, and three layers. Within each group, the networks had three neurons in the input layer, six neurons in the output layer, three to twenty-seven NHL, and three AF (tan-sig, log-sig, and linear) types. The number of architectures was determined using three factorial-type experimental designs, which reached 63, 2 187, and 50 049 combinations for G1, G2 and G3, respectively. Using MATLAB 2015a, a logical sequence was designed and implemented for constructing, training, and evaluating multilayer-perceptron-type neural networks using parallel computing techniques. The results show that HL and NHL have a statistically relevant effect on SSE, and from two hidden layers, AF also has a significant effect; thus, both AF and NHL can be evaluated to determine the optimal combination per group. Moreover, in the three study groups, it is observed that there is an inverse relationship between the number of processors and the total optimization time.

Prevalencia de patología maligna de seno en mujeres mayores de 14 años que consultaron por masa sólida palpable / Prevalence of breast malignancy in women older than forteen years who consulted for solid palpable mass

Objetivo Establecer la prevalencia de la patología maligna en pacientes con masa sólida palpable, sin diagnóstico previo de cáncer de seno en dos hospitales de Bogotá, Colombia. Materiales y métodos Estudio descriptivo retrospectivo entre marzo de 2010 y febrero de 2013 en los hospitales de San José e Infantil Universitario de San José, Bogotá D. C., Colombia. Se incluyeron mujeres mayores de 14 años que consultaron por masa sólida palpable sin diagnóstico previo de cáncer de seno, corroborada por examen físico; no se consideró ningún criterio de exclusión. Los datos se recolectaron de las historias clínicas y se llevaron a un formato creado por los investigadores. El programa estadístico utilizado fue Stata 13. Resultados Se confirmó la masa en 342 pacientes por examen clínico, en 307 pacientes con resultado de biopsia. La prevalencia de la patología maligna fue 12,2% y benigna 71,66%. Discusión La prevalencia de patología maligna por masa palpable fue menor que los datos reportados a nivel mundial, siendo el tumor más frecuente el carcinoma ductal infiltrante en un 87%, carcinoma lobulillar infiltrante en 6,4%), con estadio clínico IIA y BI-RADS 4 A (ecografía) y BI-RADS 4B (mamografía).

Objective To determine the prevalence rate of malignancy in patients with no prior breast cancer diagnosis who consulted for a solid palpable mass in two hospitals in Bogotá, Colombia. Materials and methods A descriptive retrospective study conducted between March 2010 and February 2013 at San José and Infantil Universitario de San José hospitals in Bogotá D. C., Colombia. Women 14 years or older with no prior breast cancer diagnosis who consulted for a palpable solid mass, confirmed by physical exam, were included. No exclusion criteria were considered. Data was collected from the clinical records and included in a format created by the researchers. Stata 13 was used for data analysis. Results The mass was confirmed by physical exam in 342 patients and by a biopsy in 307 patients. The prevalence rate for malignancy was 12.2% and for benign masses 71.66%. Discussion Our prevalence of breast cancer associated with a palpable mass was less than worldwide reported prevalence. The most frequent malignancy was invasive ductal carcinoma in 87% and invasive lobular carcinoma in 6.4% in stage IIA and BI-RADS 4A ultrasound category and BI-RADS 4B mammogram category.

NLRC5 shields T lymphocytes from NK-cell-mediated elimination under inflammatory conditions.

NLRC5 is a transcriptional regulator of MHC class I (MHCI), which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk, raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly, NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice, we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore, during chronic LCMV infection, the total CD8(+) T-cell population is severely decreased in these mice, a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets, having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions.

NLRC5, at the Heart of Antigen Presentation.

Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are intracellular proteins mainly involved in pathogen recognition, inflammatory responses, and cell death. Until recently, the function of the family member NLR caspase recruitment domain (CARD) containing 5 (NLRC5) has been a matter of debate. It is now clear that NLRC5 acts as a transcriptional regulator of the major-histocompatibility complex class I. In this review we detail the development of our understanding of NLRC5 function, discussing both the accepted and the controversial aspects of NLRC5 activity. We give insight into the molecular mechanisms, and the potential implications, of NLRC5 function in health and disease.

SAP modulates B cell functions in a genetic background-dependent manner.

Mutations affecting the SLAM-associated protein (SAP) are responsible for the X-linked lympho-proliferative syndrome (XLP), a severe primary immunodeficiency syndrome with disease manifestations that include fatal mononucleosis, B cell lymphoma and dysgammaglobulinemia. It is well accepted that insufficient help by SAP-/- CD4+ T cells, in particular during the germinal center reaction, is a component of dysgammaglobulinemia in XLP patients and SAP-/- animals. It is however not well understood whether in XLP patients and SAP-/- mice B cell functions are affected, even though B cells themselves do not express SAP. Here we report that B cell intrinsic responses to haptenated protein antigens are impaired in SAP-/- mice and in Rag-/- mice into which B cells derived from SAP-/- mice together with wt CD4+ T cells had been transferred. This impaired B cells functions are in part depending on the genetic background of the SAP-/- mouse, which affects B cell homeostasis. Surprisingly, stimulation with an agonistic anti-CD40 causes strong in vivo and in vitro B cell responses in SAP-/- mice. Taken together, the data demonstrate that genetic factors play an important role in the SAP-related B cell functions. The finding that anti-CD40 can in part restore impaired B cell responses in SAP-/- mice, suggests potentially novel therapeutic interventions in subsets of XLP patients.

Expansion of an osteopontin-expressing T follicular helper cell subset correlates with autoimmunity in B6.Sle1b mice and is suppressed by the H1-isoform of the Slamf6 receptor.

The costimulatory receptor Slamf6 partially controls lupus-related autoimmunity in congenic Sle1b mice; for instance, the presence of the protein isoform Slamf6-H1 in Sle1b.Slamf6-H1 mice mitigates disease. Here, we report that young Sle1b mice, but not Sle1b.Slamf6-H1 or B6 mice, contain a memory T-helper cell subset identified by ]mt]2-fold increase in expression of 17 genes, chief among which is Spp1, encoding the cytokine osteopontin (OPN). These T follicular helper (TFH) cells, including OPN(+) TFH cells, expand concomitantly with severity of the disease. By contrast, Sle1b.Slamf6-H1 or Sle1b.SAP(-)/(-) mice do not develop autoantibodies and the number of T(FH) cells is 5 times lower than in age-matched Sle1b mice. By comparing Sle1b and Sle1b.OPN(-)/(-) mice, we find that the lack of OPN expression impedes early autoantibody production. Furthermore, on the adoptive transfer of Sle1b.OPN(-)/(-) CD4(+) T cells into bm12 recipients autoantibody production and germinal center formation is reduced compared to recipients of Sle1b.OPN(+/+) CD4(+) T cells. We propose a model in which OPN provides a survival signal for a precursor T(FH) cell subset, which is a key factor in autoimmunity.

SAP expression in invariant NKT cells is required for cognate help to support B-cell responses.

One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in T, invariant natural killer T (NKT) cells and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whereas NKT cell-dependent B cell responses were absent in SAP(-/-).B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP(-/-).BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted after development in SAP(fl/fl).tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensable for noncognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell-mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.

A novel isoform of the Ly108 gene ameliorates murine lupus.

Studies of human systemic lupus erythematosus patients and of murine congenic mouse strains associate genes in a DNA segment on chromosome 1 with a genetic predisposition for this disease. The systematic analysis of lupus-prone congenic mouse strains suggests a role for two isoforms of the Ly108 receptor in the pathogenesis of the disease. In this study, we demonstrate that Ly108 is involved in the pathogenesis of lupus-related autoimmunity in mice. More importantly, we identified a third protein isoform, Ly108-H1, which is absent in two lupus-prone congenic animals. Introduction of an Ly108-H1-expressing transgene markedly diminishes T cell-dependent autoimmunity in congenic B6.Sle1b mice. Thus, an immune response-suppressing isoform of Ly108 can regulate the pathogenesis of lupus.

Auto-antibody production and glomerulonephritis in congenic Slamf1-/- and Slamf2-/- [B6.129] but not in Slamf1-/- and Slamf2-/- [BALB/c.129] mice.

Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.

Cutting edge: The adapters EAT-2A and -2B are positive regulators of CD244- and CD84-dependent NK cell functions in the C57BL/6 mouse.

EWS/FLI1-activated transcript 2 (EAT-2)A and EAT-2B are single SH2-domain proteins, which bind to phosphorylated tyrosines of signaling lymphocyte activation molecule family receptors in murine NK cells. While EAT-2 is a positive regulator in human cells, a negative regulatory role was attributed to the adapter in NK cells derived from EAT-2A-deficient 129Sv mice. To evaluate whether the genetic background or the presence of a selection marker in the mutant mice could influence the regulatory mode of these adapters, we generated EAT-2A-, EAT-2B-, and EAT-2A/B-deficient mice using C57BL/6 embryonic stem cells. We found that NK cells from EAT-2A- and EAT-2A/B-deficient mice were unable to kill tumor cells in a CD244- or CD84-dependent manner. Furthermore, EAT-2A/B positively regulate phosphorylation of Vav-1, which is known to be implicated in NK cell killing. Thus, as in humans, the EAT-2 adapters act as positive regulators of signaling lymphocyte activation molecule family receptor-specific NK cell functions in C57BL/6 mice.

Glosectomía parcial como tratamiento de macroglosia asociada a neurofibromatosis de Von Recklinghausen / Partial glossectomy as a treatment for macroglossia associated with Von Recklinghausen neurofibromatosis

Dentro de los signos de la neurofibromatosis de Von Recklinghausen se encuentra la macroglosia, la cual va a conllevar problemas de tipo foniátrico y de deglución, entre otros, para el paciente. El tratamiento de elección es la glosectomía parcial. En el presente artículo se presenta un caso referente a esto