Piotr Kraj

Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota

Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4+ Foxp3+ regulatory T (Treg) cells generated in the thymus or extrathymically by induction of naive CD4+ Foxp3− T cells. Previous studies suggested that the T-cell receptor repertoires of thymic Treg cells and induced Treg cells are biased towards self and non-self antigens, respectively, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Treg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage and favour tolerogenic presentation of antigens to naive CD4+ T cells, suggesting that intestinal homeostasis depends on microbiota-specific induced Treg cells. Here, to identify the origin and antigen-specificity of intestinal Treg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4+ Foxp3+ and CD4+ Foxp3− T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived Treg cells constitute most Treg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic Treg cells, and not induced Treg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.

Peptide specificity of thymic selection of CD4+CD25+ T cells

The CD4+CD25+ regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4+CD25+ cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4+CD25+ cells in the thymus. By comparing the development of CD4+CD25− and CD4+CD25+ cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4+CD25+ cells appear during natural selection of CD4+ T cells. The proportion of CD4+CD25+ cells in the population of CD4+ thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4+CD25+ cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4+ T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4+CD25+ thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.

Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin.

The phenotype and development of T cells from transgenic mice expressing a T cell receptor with specificity for insulin presented by the MHC class Ib molecule Qa-1(b) was investigated. Peripheral T cells from the transgenic mice express CD8 and, after activation, kill Qa-1(b)-positive lymphoid target cells in the presence of soluble insulin. Thymic selection requires expression of Qa-1(b) but not the dominant Qa-1-associated peptide, Qdm. In contrast to conventional T cells, selection is at least as efficient when the selecting ligand is expressed only on hematopoietic lineage cells as compared to expression on epithelial cells in the thymus. Our findings suggest that there is a dedicated population of Qa-1-restricted T cells that are selected by interaction with Qa-1 and that the cellular requirements for selection may differ from conventional T cells.

Foxp3-Deficient Regulatory T Cells Do Not Revert into Conventional Effector CD4+ T Cells but Constitute a Unique Cell Subset

Homeostasis in the immune system is maintained by specialized regulatory CD4+ T cells (Treg) expressing transcription factor Foxp3. According to the current paradigm, high-affinity interactions between TCRs and class II MHC-peptide complexes in thymus “instruct” developing thymocytes to up-regulate Foxp3 and become Treg cells. However, the loss or down-regulation of Foxp3 does not disrupt the development of Treg cells but abrogates their suppressor function. In this study, we show that Foxp3-deficient Treg cells in scurfy mice harboring a null mutation of the Foxp3 gene retained cellular features of Treg cells including in vitro anergy, impaired production of inflammatory cytokines, and dependence on exogenous IL-2 for proliferation and homeostatic expansion. Foxp3-deficient Treg cells expressed a low level of activation markers, did not expand relative to other CD4+ T cells, and produced IL-4 and immunomodulatory cytokines IL-10 and TGF-β when stimulated. Global gene expression profiling revealed significant similarities between Treg cells expressing and lacking Foxp3. These results argue that Foxp3 deficiency alone does not convert Treg cells into conventional effector CD4+ T cells but rather these cells constitute a distinct cell subset with unique features.

TCR Repertoire and Foxp3 Expression Define Functionally Distinct Subsets of CD4+ Regulatory T Cells

Despite extensive research efforts to characterize peripheral regulatory T (Treg) cells expressing transcription factor Foxp3, their subset complexity, phenotypic characteristics, TCR repertoire and Ag specificities remain ambiguous. In this study, we identify and define two subsets of peripheral Treg cells differing in Foxp3 expression level and TCR repertoires. Treg cells expressing a high level of Foxp3 and TCRs not used by naive CD4+ T cells present a stable suppressor phenotype and dominate the peripheral Treg population in unmanipulated mice. The second Treg subset, expressing a lower level of Foxp3 and using TCRs shared with naive CD4+ T cells constitutes a small fraction of all Treg cells in unmanipulated mice and enriches Treg population with the same Ag specificities as expressed by activated/effector T cells. This Treg subset undergoes extensive expansion during response to Ag when it becomes a major population of Ag-specific Treg cells. Thus, Treg cells expressing TCRs shared with naive CD4+ T cells have a flexible phenotype and may down-regulate Foxp3 expression which may restore immune balance at the conclusion of immune response or convert these cells to effector T cells producing inflammatory cytokines.

ParaKMeans: Implementation of a parallelized K-means algorithm suitable for general laboratory use

BackgroundDuring the last decade, the use of microarrays to assess the transcriptome of many biological systems has generated an enormous amount of data. A common technique used to organize and analyze microarray data is to perform cluster analysis. While many clustering algorithms have been developed, they all suffer a significant decrease in computational performance as the size of the dataset being analyzed becomes very large. For example, clustering 10000 genes from an experiment containing 200 microarrays can be quite time consuming and challenging on a desktop PC. One solution to the scalability problem of clustering algorithms is to distribute or parallelize the algorithm across multiple computers.ResultsThe software described in this paper is a high performance multithreaded application that implements a parallelized version of the K-means Clustering algorithm. Most parallel processing applications are not accessible to the general public and require specialized software libraries (e.g. MPI) and specialized hardware configurations. The parallel nature of the application comes from the use of a web service to perform the distance calculations and cluster assignments. Here we show our parallel implementation provides significant performance gains over a wide range of datasets using as little as seven nodes. The software was written in C# and was designed in a modular fashion to provide both deployment flexibility as well as flexibility in the user interface.ConclusionParaKMeans was designed to provide the general scientific community with an easy and manageable client-server application that can be installed on a wide variety of Windows operating systems.

αβTCRs Differ in the Degree of Their Specificity for the Positively Selecting MHC/Peptide Ligand

We have tested the peptide specificity of positive selection using three transgenic αβTCRs, originally selected on class II MHC (Ab) covalently bound with one peptide Eα (52–68) (Ep). The transgenic TCR specific for the cytochrome c-derived (43–58) peptide was selected on Ab bound with different arrays of endogenous peptides or the analogue of Ep covalently bound to Ab, but not on the original AbEp complex. In contrast, transgenic TCRs specific for two different analogues of the Ep peptide and Ab did not mature as CD4+ T cells in various thymic environments, including the AbEpIi− mice. These results show that TCRs can be promiscuous or specific for the selecting MHC/peptide complex, and suggest that in mice described in this study transgenic expression of the TCR changes the original requirements for the positively selecting MHC/peptide complex. Future studies will determine whether the latter phenomenon is general or specific for this system.

Connexin 43 Signaling Enhances the Generation of Foxp3+ Regulatory T Cells

Despite their importance for the functioning of the immune system, thymic development and peripheral maintenance of Foxp3+ regulatory T (TR) cells are poorly understood. We have found that connexin 43 (Cx43), expressed by thymic TR cells progenitors, supports TR development. Mice with deletion of the Cx43 gene induced in T cells produce only few TR cells and had increased proportion of activated T cells in the lymph nodes, suggesting impaired peripheral tolerance. Reduction of the TR cell numbers was accompanied by increased presence of CD4+CD25+GITR+Foxp3− T cells, which did not produce inflammatory cytokines and lost suppressor function. These results strongly argue that we have discovered a novel signaling pathway, controlled by Cx43, that enhances the generation of TR cells. We propose that a possible mechanism of Cx43 activity is by regulating Foxp3 expression in TR lineage cells.

Systematic evaluation of 640 FDA drugs for their effect on CD4⁺Foxp3⁺ regulatory T cells using a novel cell-based high throughput screening assay.

Regulatory T cells (Treg), which play a pivotal role in maintaining immune homeostasis by suppressing the proliferation of effector T cells, have great therapeutic potential for autoimmune diseases and transplantation. However, progress on their clinical application has been hampered by the lack of high throughput screening (HTS) strategies for the systematic and rapid evaluation of existing drugs and the identification of novel drug candidates. In this report, we present an innovative in vitro HTS assay using CD4⁺ T cells from Foxp3-GFP transgenic mice that specifically express the GFP signal in Foxp3⁺ Treg cells detectable by FACS analysis in a high throughput manner. Systematic evaluation of 640 FDA-approved drugs revealed that 70 drugs increased the number of Treg cells with suppression function only in the presence of TGFβ, 75 drugs increased Treg numbers even in the absence of TGFβ, and 32 drugs increased Treg numbers synergistically with TGFβ. The identified Treg-promoting drugs include those previously known to induce Treg (rapamycin and retinoic acid), statins, glucocorticoids and drugs in many other categories. Furthermore, Treg cells cultured with the identified drugs possess surface and intracellular markers characteristic of natural Treg cells and possess suppressive function. These results suggest that this Treg HTS assay can be used to screen compound libraries to identify novel chemical entities for Treg-based immune therapies.

Intratumoral Convergence of the TCR Repertoires of Effector and Foxp3+ CD4+ T cells

The presence of Foxp3+ regulatory CD4+ T cells in tumor lesions is considered one of the major causes of ineffective immune response in cancer. It is not clear whether intratumoral Treg cells represent Treg cells pre-existing in healthy mice, or arise from tumor-specific effector CD4+ T cells and thus representing adaptive Treg cells. The generation of Treg population in tumors could be further complicated by recent evidence showing that both in humans and mice the peripheral population of Treg cells is heterogenous and consists of subsets which may differentially respond to tumor-derived antigens. We have studied Treg cells in cancer in experimental mice that express naturally selected, polyclonal repertoire of CD4+ T cells and which preserve the heterogeneity of the Treg population. The majority of Treg cells present in healthy mice maintained a stable suppressor phenotype, expressed high level of Foxp3 and an exclusive set of TCRs not used by naive CD4+ T cells. A small Treg subset, utilized TCRs shared with effector T cells and expressed a lower level of Foxp3. We show that response to tumor-derived antigens induced efficient clonal recruitment and expansion of antigen-specific effector and Treg cells. However, the population of Treg cells in tumors was dominated by cells expressing TCRs shared with effector CD4+ T cells. In contrast, Treg cells expressing an exclusive set of TCRs, that dominate in healthy mice, accounted for only a small fraction of all Treg cells in tumor lesions. Our results suggest that the Treg repertoire in tumors is generated by conversion of effector CD4+ T cells or expansion of a minor subset of Treg cells. In conclusion, successful cancer immunotherapy may depend on the ability to block upregulation of Foxp3 in effector CD4+ T cells and/or selectively inhibiting the expansion of a minor Treg subset.