Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice.

The use of flow cytometry in mice is constrained by several factors, including the limited availability of mouse-specific antibodies and the need to work with small volumes of peripheral blood. This is particularly challenging for longitudinal studies, as serial blood samples should not exceed 10% of the total blood volume in mice. To address this, we have developed two novel flow cytometry panels designed to extensively analyze immune cell populations in mice during longitudinal studies, using only 50 µL of peripheral blood per panel. Additionally, a third panel has been designed to conduct a more detailed analysis of cytotoxic and inhibitory markers at the end point. These panels have been validated on a lipopolysaccharide (LPS)-induced lung inflammation model. Two experiments were conducted to 1) validate the panels' sensitivity to immune challenges (n=12) and 2) to assess intrinsic variability of measurements (n=5). In both experiments, we collected 50 µL of peripheral blood for each cytometry panel from the maxillary venous sinus. All antibodies were titrated to identify the optimal concentration that maximized the signal from the positive population while minimizing the signal from the negative population. Samples were processed within 1 hour of collection using a MACSQuant Analyzer 16 cytometer. Our results demonstrate that these immunological panels are sensitive enough to detect changes in peripheral blood after LPS induction. Moreover, our findings help determine the sample size needed based on the immune population variability. In conclusion, the panels we have designed enable a comprehensive analysis of the murine immune system with a low blood volume requirement, enabling the measure of both absolute values and relative percentages effectively. This approach provides a robust platform for longitudinal studies in mice and can be used to uncover significant insights into immune responses.

Multidimensional analysis of immune cells from COVID-19 patients identified cell subsets associated with the severity at hospital admission.

BACKGROUND: SARS-CoV-2 emerged as a new coronavirus causing COVID-19, and it has been responsible for more than 760 million cases and 6.8 million deaths worldwide until March 2023. Although infected individuals could be asymptomatic, other patients presented heterogeneity and a wide range of symptoms. Therefore, identifying those infected individuals and being able to classify them according to their expected severity could help target health efforts more effectively. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we wanted to develop a machine learning model to predict those who will develop severe disease at the moment of hospital admission. We recruited 75 individuals and analysed innate and adaptive immune system subsets by flow cytometry. Also, we collected clinical and biochemical information. The objective of the study was to leverage machine learning techniques to identify clinical features associated with disease severity progression. Additionally, the study sought to elucidate the specific cellular subsets involved in the disease following the onset of symptoms. Among the several machine learning models tested, we found that the Elastic Net model was the better to predict the severity score according to a modified WHO classification. This model was able to predict the severity score of 72 out of 75 individuals. Besides, all the machine learning models revealed that CD38+ Treg and CD16+ CD56neg HLA-DR+ NK cells were highly correlated with the severity. CONCLUSIONS/SIGNIFICANCE: The Elastic Net model could stratify the uninfected individuals and the COVID-19 patients from asymptomatic to severe COVID-19 patients. On the other hand, these cellular subsets presented here could help to understand better the induction and progression of the symptoms in COVID-19 individuals.

Cellular and Humoral Responses Follow-up for 8 Months after Vaccination with mRNA-Based Anti-SARS-CoV-2 Vaccines.

Vaccination against SARS-CoV-2 has become the main method of reducing mortality and severity of COVID-19. This work aims to study the evolution of the cellular and humoral responses conferred by two mRNA vaccines after two doses against SARS-CoV-2. On days 30 and 240 after the second dose of both vaccines, the anti-S antibodies in plasma were evaluated from 82 volunteers vaccinated with BNT162b2 and 68 vaccinated with mRNA-1273. Peripheral blood was stimulated with peptides encompassing the entire SARS-CoV-2 Spike sequence. IgG Anti-S antibodies (humoral) were quantified on plasma, and inflammatory cytokines (cellular) were measured after stimulation. We observed a higher response (both humoral and cellular) with the mRNA-1273 vaccine. Stratifying by age and gender, differences between vaccines were observed, especially in women under 48 and men over 48 years old. Therefore, this work could help to set up a vaccination strategy that could be applied to confer maximum immunity.

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy.

Due to their suppressive capacity, the adoptive transfer of regulatory T cells (Treg) has acquired a growing interest in controlling exacerbated inflammatory responses. Limited Treg recovery and reduced quality remain the main obstacles in most current protocols where differentiated Treg are obtained from adult peripheral blood. An alternate Treg source is umbilical cord blood, a promising source of Treg cells due to the higher frequency of naïve Treg and lower frequency of memory T cells present in the fetus' blood. However, the Treg number isolated from cord blood remains limiting. Human thymuses routinely discarded during pediatric cardiac surgeries to access the retrosternal operative field has been recently proposed as a novel source of Treg for cellular therapy. This strategy overcomes the main limitations of current Treg sources, allowing the obtention of very high numbers of undifferentiated Treg. We have developed a novel good manufacturing practice (GMP) protocol to obtain large Treg amounts, with very high purity and suppressive capacity, from the pediatric thymus (named hereafter thyTreg). The total amount of thyTreg obtained at the end of the procedure, after a short-term culture of 7 days, reach an average of 1,757 x106 (range 50 x 106 - 13,649 x 106) cells from a single thymus. The thyTreg product obtained with our protocol shows very high viability (mean 93.25%; range 83.35% - 97.97%), very high purity (mean 92.89%; range 70.10% - 98.41% of CD25+FOXP3+ cells), stability under proinflammatory conditions and a very high suppressive capacity (inhibiting in more than 75% the proliferation of activated CD4+ and CD8+ T cells in vitro at a thyTreg:responder cells ratio of 1:1). Our thyTreg product has been approved by the Spanish Drug Agency (AEMPS) to be administered as cell therapy. We are recruiting patients in the first-in-human phase I/II clinical trial worldwide that evaluates the safety, feasibility, and efficacy of autologous thyTreg administration in children undergoing heart transplantation (NCT04924491). The high quality and amount of thyTreg and the differential features of the final product obtained with our protocol allow preparing hundreds of doses from a single thymus with improved therapeutic properties, which can be cryopreserved and could open the possibility of an "off-the-shelf" allogeneic use in another individual.

ABO blood group is involved in the quality of the specific immune response anti-SARS-CoV-2.

Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12-305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the spike (S), nucleocapsid (N) and membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that O-group individuals presented significantly lower frequencies of specific CD4+ T-cell responses against Pep-M than non O-group individuals. The non O-group subjects also needed longer to clear the virus, and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors might determine the sustainability of the body's defenses, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity.

An international virtual hackathon to build tools for the analysis of structural variants within species ranging from coronaviruses to vertebrates.

In October 2020, 62 scientists from nine nations worked together remotely in the Second Baylor College of Medicine & DNAnexus hackathon, focusing on different related topics on Structural Variation, Pan-genomes, and SARS-CoV-2 related research.   The overarching focus was to assess the current status of the field and identify the remaining challenges. Furthermore, how to combine the strengths of the different interests to drive research and method development forward. Over the four days, eight groups each designed and developed new open-source methods to improve the identification and analysis of variations among species, including humans and SARS-CoV-2. These included improvements in SV calling, genotyping, annotations and filtering. Together with advancements in benchmarking existing methods. Furthermore, groups focused on the diversity of SARS-CoV-2. Daily discussion summary and methods are available publicly at  https://github.com/collaborativebioinformatics provides valuable insights for both participants and the research community.

Ectopic FOXP3 Expression in Combination with TGF-ß1 and IL-2 Stimulation Generates Limited Suppressive Function in Human Primary Activated Thymocytes Ex Vivo.

Regulatory T cells (Tregs), which are characterized by the expression of the transcription factor forkhead box P3 (FOXP3), are the main immune cells that induce tolerance and are regulators of immune homeostasis. Natural Treg cells (nTregs), described as CD4+CD25+FOXP3+, are generated in the thymus via activation and cytokine signaling. Transforming growth factor beta type 1 (TGF-ß1) is pivotal to the generation of the nTreg lineage, its maintenance in the thymus, and to generating induced Treg cells (iTregs) in the periphery or in vitro arising from conventional T cells (Tconvs). Here, we tested whether TGF-ß1 treatment, associated with interleukin-2 (IL-2) and CD3/CD28 stimulation, could generate functional Treg-like cells from human thymocytes in vitro, as it does from Tconvs. Additionally, we genetically manipulated the cells for ectopic FOXP3 expression, along with the TGF-ß1 treatment. We demonstrated that TGF-ß1 and ectopic FOXP3, combined with IL-2 and through CD3/CD28 activation, transformed human thymocytes into cells that expressed high levels of Treg-associated markers. However, these cells also presented a lack of homogeneous suppressive function and an unstable proinflammatory cytokine profile. Therefore, thymocyte-derived cells, activated with the same stimuli as Tconvs, were not an appropriate alternative for inducing cells with a Treg-like phenotype and function.

The Presence of a Marked Imbalance Between Regulatory T Cells and Effector T Cells Reveals That Tolerance Mechanisms Could Be Compromised in Heart Transplant Children.

Regulatory T cells (Treg) are crucial for the induction and maintenance of graft tolerance. In pediatric heart transplant procedures, the thymus is routinely excised, removing the primary source of T-cell replenishment. Consequently, thymectomy joined to the effects of immunosuppression on the T-cell compartment may have a detrimental impact on Treg values, compromising the intrinsic tolerance mechanisms and the protective role of Treg preventing graft rejection in heart transplant children. METHODS: A prospective study including 7 heart transplant children was performed, and immune cell populations were evaluated periodically in fresh peripheral blood at different time points before and up to 3 y posttransplant. RESULTS: Treg counts decreased significantly from the seventh-month posttransplant. Furthermore, there was a significant increase in effector memory and terminally differentiated effector memory T cells coinciding with the fall of Treg counts. The Treg/Teffector ratio, a valuable marker of the tolerance/rejection balance, reached values around 90% lower than pretransplant values. Additionally, a negative correlation between Treg count and T effector frequency was observed. Particularly, when Treg count decreases below 50 or 75 cells/µL in the patients, the increase in the frequency of T effector CD4+ and CD8+, respectively, experiences a tipping point, and the proportion of T-effector cells increases dramatically. CONCLUSIONS: These results reveal that interventions employed in pediatric heart transplantation (immunosuppression and thymectomy) could induce, as an inevitable consequence, a dysregulation in the immunologic status characterized by a marked imbalance between Treg and T effector, which could jeopardize the preservation of tolerance during the period with the higher incidence of acute rejection.

Comprehensive Flow Cytometry Profiling of the Immune System in COVID-19 Convalescent Individuals.

SARS-CoV-2 has infected more than 200 million people worldwide, with more than 4 million associated deaths. Although more than 80% of infected people develop asymptomatic or mild COVID-19, SARS-CoV-2 can induce a profound dysregulation of the immune system. Therefore, it is important to investigate whether clinically recovered individuals present immune sequelae. The potential presence of a long-term dysregulation of the immune system could constitute a risk factor for re-infection and the development of other pathologies. Here, we performed a deep analysis of the immune system in 35 COVID-19 recovered individuals previously infected with SARS-CoV-2 compared to 16 healthy donors, by flow cytometry. Samples from COVID-19 individuals were analysed from 12 days to 305 days post-infection. We observed that, 10 months post-infection, recovered COVID-19 patients presented alterations in the values of some T-cell, B-cell, and innate cell subsets compared to healthy controls. Moreover, we found in recovered COVID-19 individuals increased levels of circulating follicular helper type 1 (cTfh1), plasmablast/plasma cells, and follicular dendritic cells (foDC), which could indicate that the Tfh-B-foDC axis might be functional to produce specific immunoglobulins 10 months post-infection. The presence of this axis and the immune system alterations could constitute prognosis markers and could play an important role in potential re-infection or the presence of long-term symptoms in some individuals.

Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline.

Feather mites are among the most common and diverse ectosymbionts of birds, yet basic questions such as the nature of their relationship remain largely unanswered. One reason for feather mites being understudied is that their morphological identification is often virtually impossible when using female or young individuals. Even for adult male specimens this task is tedious and requires advanced taxonomic expertise, thus hampering large-scale studies. In addition, molecular-based methods are challenging because the low DNA amounts usually obtained from these tiny mites do not reach the levels required for high-throughput sequencing. This work aims to overcome these issues by using a DNA metabarcoding approach to accurately identify and quantify the feather mite species present in a sample. DNA metabarcoding is a widely used molecular technique that takes advantage of high-throughput sequencing methodologies to assign the taxonomic identity to all the organisms present in a complex sample (i.e., a sample made up of multiple specimens that are hard or impossible to individualise). We present a high-throughput method for feather mite identification using a fragment of the COI gene as marker and Illumina Miseq technology. We tested this method by performing two experiments plus a field test over a total of 11,861 individual mites (5360 of which were also morphologically identified). In the first experiment, we tested the probability of detecting a single feather mite in a heterogeneous pool of non-conspecific individuals. In the second experiment, we made 2 × 2 combinations of species and studied the relationship between the proportion of individuals of a given species in a sample and the proportion of sequences retrieved to test whether DNA metabarcoding can reliably quantify the relative abundance of mites in a sample. Here we also tested the efficacy of degenerate primers (i.e., a mixture of similar primers that differ in one or several bases that are designed to increase the chance of annealing) and investigated the relationship between the number of mismatches and PCR success. Finally, we applied our DNA metabarcoding pipeline to a total of 6501 unidentified and unsorted feather mite individuals sampled from 380 European passerine birds belonging to 10 bird species (field test). Our results show that this proposed pipeline is suitable for correct identification and quantitative estimation of the relative abundance of feather mite species in complex samples, especially when dealing with a moderate number (> 30) of individuals per sample.

The complete mitochondrial genome of the feather mite Trouessartia rubecula Jablonska, 1968 (Astigmata: Analgoidea: Trouessartiidae).

We assembled and annotated the complete mitochondrial genome of Trouessartia rubecula, the first feather mite complete mitochondrial genome from the largest feather mite superfamily Analgoidea (ca. 1150 spp). The mitogenome was composed of 13 protein, 17 tRNA, and 2 rRNA-coding genes and was 14,125 bp in length.

The plastid ancestor originated among one of the major cyanobacterial lineages.

The primary endosymbiotic origin of chloroplasts is now well established but the identification of the present cyanobacteria most closely related to the plastid ancestor remains debated. We analyse the evolutionary trajectory of a subset of highly conserved cyanobacterial proteins (core) along the plastid lineage, those which were not lost after the endosymbiosis. We concatenate the sequences of 33 cyanobacterial core proteins that share a congruent evolutionary history, with their eukaryotic counterparts to reconstruct their phylogeny using sophisticated evolutionary models. We perform an independent reconstruction using concatenated 16S and 23S rRNA sequences. These complementary approaches converge to a plastid origin occurring during the divergence of one of the major cyanobacterial lineages that include N2-fixing filamentous cyanobacteria and species able to differentiate heterocysts.

In vivo expression of a Cicer arietinum beta-galactosidase in potato tubers leads to a reduction of the galactan side-chains in cell wall pectin.

We report the generation of Solanum tuberosum transformants expressing Cicer arietinum betaIII-Gal. betaIII-Gal is a beta-galactosidase able to degrade cell wall pectins during cell wall loosening that occurs prior to cell elongation. cDNA corresponding to the gene encoding this protein was identified among several chickpea beta-galactosidase cDNAs, and named CanBGal-3. CanBGal-3 cDNA was expressed in potato under the control of the granule-bound starch synthase promoter. Three betaIII-Gal transformants with varying levels of expression were chosen for further analysis. The transgenic plants displayed no significant altered phenotype compared to the wild type. However, beta-galactanase and beta-galactosidase activities were increased in the transgenic tuber cell walls and this affected the potato tuber pectins. A reduction in the galactosyl content of up to 50% compared to the wild type was observed in the most extreme transformant, indicating a reduction of 1,4-beta-galactan side-chains, as revealed by analysis with LM5 specific antibodies. Our results confirm the notion that the pectin-degrading activity of chickpea betaIII-Gal reported in vitro also occurs in vivo and in other plants, and confirm the involvement of betaIII-Gal in the cell wall autolysis process. An increase in the homogalacturonan content of transgenic tuber cell walls was also observed by Fourier transform infrared spectroscopy (FTIR) analysis.

Cloning of a Cicer arietinum beta-galactosidase with pectin-degrading function.

The cDNA clone (CanBGal-3) encoding a cell wall pectin-degrading beta-galactosidase (beta III-Gal) from Cicer arietinum L. cv. Castellana has been identified. The identification was carried out by comparing the deduced amino acid sequences of several isolated chickpea beta-galactosidase clones with the purified beta III-Gal protein sequence. The expression pattern of the gene corresponding to CanBGal-3 was in concordance with the fluctuations of the enzyme beta III-Gal in different seedling organs, being specific to elongating organs such as epicotyls and roots. Transformation of Solanum tuberosum plants with the chickpea CanBGal-3 clone indicated that the beta-galactosidase encoded by this clone is a pectin-degrading enzyme. The authors propose an important role for chickpea beta III-Gal in pectin degradation in cell walls of vegetative organs such as epicotyls and roots. The degradation of galactan carried out by this enzyme may determine structural changes and affect cell wall porosity. It is suggested that the increase in the size of cell wall pores could permit access of other cell wall-modifying enzymes to their substrate.

A seedling specific vegetative lectin gene is related to development in Cicer arietinum.

Plant lectins are a group of glycoproteins with the ability to recognize and bind carbohydrate ligands. Seed lectins function as storage and defense proteins, but the specific function of vegetative lectins is uncertain. In this paper we describe the characterization of a clone, CanVLEC, encoding a vegetative lectin from chickpea (Cicer arietinum L. cv. Castellana). The expression of the CanVLEC gene was specific in seedlings, mostly in hooks and elongating epicotyls, and no expression was detected in adult plants. The level of chickpea vegetative lectin transcripts in epicotyls decreased through the epicotyl growth suggesting a relationship to development. Treatment with indoleacetic acid (IAA) and brassinolides (BR), hormones that promoted elongation in chickpea epicotyl, increased the level of CanVLEC mRNA, supporting a relationship to growth. CanVLEC is drastically down regulated by water deficit ruling out its possible involvement in plant response to water stress, unlike other vegetative lectins. CanVLEC protein may be targeted to an extracellular location owing to the presence of a signal peptide.