Jose Faro

Regulation of the Germinal Center Reaction by Foxp3+ Follicular Regulatory T Cells

Follicular helper T (TFH) cells participate in humoral responses providing selection signals to germinal center B cells. Recently, expression of CXCR5, PD-1, and the transcription factor Bcl-6 has allowed the identification of TFH cells. We found that a proportion of follicular T cells, with phenotypic characteristics of TFH cells and expressing Foxp3, are recruited during the course of a germinal center (GC) reaction. These Foxp3+ cells derive from natural regulatory T cells. To establish the in vivo physiologic importance of Foxp3+ follicular T cells, we used CXCR5-deficient Foxp3+ cells, which do not have access to the follicular region. Adoptive cell transfers of CXCR5-deficient Foxp3+ cells have shown that Foxp3+ follicular T cells are important regulators of the GC reaction following immunization with a thymus-dependent Ag. Our in vivo data show that Foxp3+ follicular T cells can limit the magnitude of the GC reaction and also the amount of secreted Ag-specific IgM, IgG1, IgG2b, and IgA. Therefore, Foxp3+ follicular regulatory T cells appear to combine characteristics of TFH and regulatory T cells for the control of humoral immune responses.

Assessing Methods for Blood Cell Cytotoxic Responses to Inorganic Nanoparticles and Nanoparticle Aggregates

Inorganic nanoparticles (NPs) show great potential for medicinal therapy. However, biocompatibility studies are essential to determine if they are safe. Here, five different NPs are compared for their cytotoxicity, internalization, aggregation in medium, and reactive oxygen species (ROS) production, using tumoral and normal human blood cells. Differences depending on the cell type are analyzed, and no direct correlation between ROS production and cell toxicity is found. Results are discussed with the aim of standardizing the procedures for the evaluation of the toxicity.

When three is not a crowd : a Crossregulation Model of the dynamics and repertoire selection of regulatory CD4+ T cells

Summary:  Regulatory CD4+ T cells, enriched in the CD25 pool of healthy individuals, mediate natural tolerance and prevent autoimmune diseases. Despite their fundamental and potential clinical significance, regulatory T (TR) cells have not yet been incorporated in a coherent theory of the immune system. This article reviews experimental evidence and theoretical arguments supporting a model of TR cell dynamics, uncovering some of its most relevant biological implications. According to this model, the persistence and expansion of TR cell populations depend strictly on specific interactions they make with antigen‐presenting cells (APCs) and conventional effector T (TE) cells. This three‐partner crossregulation imposes that TR cells feed on the specific autoimmune activities they suppress, with implications ranging from their interactions with other cells to their repertoire selection in the periphery and in the thymus, and to the relationship between these cells and the innate immune system. These implications stem from the basic prediction that the peripheral dynamics sort the CD4+ T‐cell repertoire into two subsets: a less diverse set of small clones of autoreactive effector and regulatory cells that regulate each other’s growth, and a more diverse set of barely autoreactive TE cell clones, whose expansion is limited only by APC availability. It is argued that such partitioning of the repertoire sets the ground for self–non‐self discrimination.

An approximation for prey-predator models with time delay

Abstract A prototype prey-predator (P-D) model in which the effective size of the predator population interacting with its prey follows an instantaneous time-delay τ regarding its total size is considered here. A simplified model was derived after substituting the approximation D(t − τ) ≈ D(t) − τ D (t) into the above time-delay model. In order to assess the reliability of the simplified model, we performed a comparative study of both models under a wide range of parameter values, focusing on the effect of τ on two issues: (i) the boundary (in parameter space) between the regions leading either to stable fixed points characteristic of the non-delay model, both the boundary and the periods and amplitudes obtained for the time-delay model can be fairly approximated by the corresponding results for the simplified model.

T follicular helper and T follicular regulatory cells have different TCR specificity

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity.

The Impact of Thymic Antigen Diversity on the Size of the Selected T Cell Repertoire

The TCR repertoire of a normal animal is shaped in the thymus by ligand-specific positive- and negative-selection events. These processes are believed to be determined at the single-cell level primarily by the affinity of the TCR-ligand interactions. The relationships among all the variables involved are still unknown due to the complexity of the interactions and the lack of quantitative analysis of those parameters. In this study, we developed a quantitative model of thymic selection that provides estimates of the fractions of positively and negatively selected thymocytes in the cortex and in the medulla, as well as upper-bound ranges for the number of selecting ligands required for the generation of a normal diverse TCR repertoire. Fitting the model to current estimates of positive- and negative-selected thymocytes leads to specific predictions. The results indicate the following: 1) the bulk of thymocyte death takes place in the cortex, and it is due to neglect; 2) the probability of a thymocyte to be negatively selected in the cortex is at least 10-fold lower than in the medulla; 3) <60 ligands are involved in cortical positive selection; and 4) negative selection in the medulla is constrained by a large diversity of selecting ligands on medullary APCs.

An automated algorithm for extracting functional immunologic V-genes from genomes in jawed vertebrates

Variable (V) domains of immunoglobulins (Ig) and T cell receptors (TCR) are generated from genomic V gene segments (V-genes). At present, such V-genes have been annotated only within the genome of a few species. We have developed a bioinformatics tool that accelerates the task of identifying functional V-genes from genome datasets. Automated recognition is accomplished by recognizing key V-gene signatures, such as recombination signal sequences, size of the exon region, and position of amino acid motifs within the translated exon. This algorithm also classifies extracted V-genes into either TCR or Ig loci. We describe the implementation of the algorithm and validate its accuracy by comparing V-genes identified from the human and mouse genomes with known V-gene annotations documented and available in public repositories. The advantages and utility of the algorithm are illustrated by using it to identify functional V-genes in the rat genome, where V-gene annotation is still incomplete. This allowed us to perform a comparative human–rodent phylogenetic analysis based on V-genes that supports the hypothesis that distinct evolutionary pressures shape the TCRs and Igs V-gene repertoires. Our program, together with a user graphical interface, is available as open-source software, downloadable at http://code.google.com/p/vgenextract/.

Genomic V exons from whole genome shotgun data in reptiles

Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

Re-evaluating the recycling hypothesis in the germinal centre.

Mathematical models have been used to study different aspects of the germinal centre reaction, in particular, affinity maturation of antibodies and the hypothesis of recycling. So far, interpretation of several theoretical and experimental results has pointed to the existence of recycling. However, theoretical models have seldom been compared with experimental data from specific immune responses and the potential relevance of recycling in the germinal centre is still an open problem. In this article, we propose a model without recycling that takes into account selection mechanisms that were previously uncovered experimentally. We apply the model to several experimental systems that use different Ag and compare the results with experimental data of affinity maturation whenever available. The results obtained for a primary immune response to the hapten (4‐hydroxy‐3‐nitrophenyl)‐acetyl show that recycling is not a necessary mechanism to achieve the level of affinity maturation observed in germinal centre reactions. Similar levels of affinity maturation are obtained for other responses, although for antibodies involving several affinity‐enhancing mutations the affinity maturation obtained with the model is much lower. Interpretation of these results and consequences towards the concept of recycling are discussed.

Modelling Two Possible Mechanisms for the Regulation of the Germinal Center Dynamics

Research on the germinal center has tried to unravel the mechanisms that control its dynamics. In this study we focus on the termination of the germinal center reaction, which is still an open problem. We propose two hypothetical biological mechanisms that may be responsible for the control of germinal center dynamics and analyze them through mathematical models. The first one is based on the differentiation of follicular dendritic cells and/or T cells. Interaction of these cells in the differentiated state with germinal center B cells would promote B cell differentiation into memory B cells and Ab-forming cells, ending the germinal center reaction. The second mechanism applies only to a scenario without recycling and consists of the decay of a hypothetical proliferation signal for centroblasts that limits the number of cell divisions. Each of the models makes predictions that can be experimentally tested.