Integrating Heterogeneous Odor Response Data into a Common Response Model: A DoOR to the Complete Olfactome

We have developed a new computational framework for merging odor response data sets from heterogeneous studies, creating a consensus metadatabase, the database of odor responses (DoOR). As a result, we obtained a functional atlas of all available odor responses in Drosophila melanogaster. Both the program and the data set are freely accessible and downloadable on the Internet (http://neuro.uni-konstanz.de/DoOR). The procedure can be adapted to other species, thus creating a family of “olfactomes” in the near future. Drosophila melanogaster was chosen because of all species this one is closest to having the complete olfactome characterized, with the highest number of deorphanized receptors available. The database guarantees long-term stability (by offering time-stamped, downloadable versions), up-to-date accuracy (by including new data sets as soon as they are published), and portability (for other species). We hope that this comprehensive repository of odor response profiles will be useful to the olfactory community and to computational neuroscientists alike

DoOR.functions v2.0.0

<p>A comprehensive update to data and functions of the DoOR project. Please see the publication for more details: http://dx.doi.org/10.1038/srep21841</p

T_reg cells lacking TR2 are functional.

<p>(A) <i>In vitro</i> suppression assay: sorted conventional CD45.1⁺CD4⁺ T cells were stimulated with anti-CD3 (2 µg/ml) and cocultured with sorted WT T_reg cells or tam-iCD4TR2 T_reg cells (isolated 14 d p.a.) at various ratios. Thymidine was added for the last 24 h of culture. Analysis was performed after 96 h. Percent suppression as mean ± SD (analysed in two independent experiments). (B) <i>In vitro</i> suppression assay: sorted conventional CD45.1⁺CD4⁺ T cells were labelled with CFSE, stimulated with anti-CD3 (2 µg/ml), and cocultured with sorted wt T_reg cells or tam-iCD4TR2 T_reg cells (isolated 14 d p.a.) at various ratios. FACS analysis was performed after 96 h. These data are representative results of two independent experiments. (C and D) <i>In vivo</i> suppression assay: Development of colitis in Rag1^−/− mice after transfer of conventional CD4⁺ T cells alone or in combination with tam-iCD4TR2 (mice treated for 5 d, cells isolated 1 wk p.a.) T_reg cells or iCD4TR2 T_reg cells. Change in body weight after 8 wk posttransfer (mean, 3 mice per group, representative data of two independent experiments). (D) Representative micrographs of H&E-stained small intestine sections from <i>in vivo</i> suppression experiments isolated from Rag1^−/− mice 8 wk after transfer of the indicated cells. Scoring of colitis severity according to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001674#pbio.1001674-Asseman1" target="_blank">[60]</a>. (E) Criss-cross <i>in vitro</i> suppression assay: sorted conventional tam-iCD4TR2 and WT T cells were cocultured with sorted tam-iCD4TR2 and wt T_reg cells at various ratios. Analysis was performed after 96 h (representative data of two independent experiments). (F) Development of colitis in Rag1^−/− mice after adoptive transfer of conventional tam-iCD4TR2 and wt T cells alone or in combination with tam-iCD4TR2 T_reg cells. Change in body weight after 8 wk posttransfer (mean ± SEM, 3 mice per group, representative data of two independent experiments).</p

Deregulated proliferation control upon removal of TR2.

<p>(A) Flow cytometric analysis of CD69 expression by CD4⁺ splenic T cells isolated 2 wk p.a. These data are representative results of three independent experiments. (B) Analysis of sensitivity to activation through measurement of proliferation. Sorted CD4⁺ T cells were cultured for 72 h and stimulated with different anti-CD3 concentrations. Thymidine was added for the last 24 h of culture (mean ± SEM, 4 mice per group, analysed in two independent experiments). (C) Flow cytometric analysis of cytoplasmic calcium by ratiometric measurement of Indo-1–labelled cells from tam-iCD4TR2 and control mice. TCR crosslinking was performed after 15 s. On the left mean ratio of the baseline (representative data of two independent experiments). (D) Flow cytometric analysis of IL-2, CD25, and CD122 expression by splenic CD4⁺ T cells isolated from tam-iCD4TR2 and control mice. These data are representative results of two independent experiments. (E and F) Proliferation analysis of sorted CD4⁺ T cells cultured for 72 h with anti-CD3 (0.6 µg/ml) and anti-CD25 (PC61) (E) or indicated cytokines (F). Thymidine was added for the last 24 h of culture (mean ± SEM, 4 mice per group, analysed in two independent experiments).</p

Increased proliferation of regulatory T cells upon removal of TR2.

<p>(A) The percentage of T_reg cells (left panel) and number of T_reg cells (right panel) in the spleen of tam-iCD4TR2 and control mice at indicated time points (mean ± SEM, 9 mice per group, analysed in two independent experiments). (B) The percentage of T_reg cells in the indicated organs of tam-iCD4TR2 and control mice (mean ± SEM, 5 mice per group, analysed in two independent experiments). (C) The percentage of T_reg cells in the spleens of thymectomised tam-iCD4TR2 and control mice 20 wk p.a. (mean ± SEM, 9 mice per group, analysed in two independent experiments). (D) The percentage of T_reg (left panel) and absolute number of Treg cells (right panel) within the LN CD4⁺ T cells of the indicated CD45.1⁺ or CD45.2⁺ bone marrow–derived cells (experiment described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001674#pbio-1001674-g005" target="_blank">Figure 5E</a>, mean ± SEM, 10 mice per group, analysed in three independent experiments). (E) The percentage of BrdU⁺ T_reg cells isolated from LN (experiment described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001674#pbio-1001674-g005" target="_blank">Figure 5E</a>, mean ± SEM, 10 mice per group, analysed in three independent experiments). (F) Flow cytometric analysis of Nrp-1 and Foxp3 expression by CD4⁺ T cells (left panel) and the percentage of Nrp-1⁺ and Nrp-1⁻ T_reg cells within the LN CD4⁺ T cells of tam-iCD4TR2 and control mice 2 wk p.a. (right panel) (mean ± SEM, 9 mice per group, analysed in two independent experiments). (G) Flow cytometric analysis of Ki-67 expression by Nrp-1⁺ T_reg cells (left panel) and the percentage of Ki-67⁺Nrp-1⁺ T_reg cells within the LN CD4⁺ T cells of tam-iCD4TR2 and control mice 2 wk p.a. (right panel) (mean ± SEM, 9 mice per group, analysed in two independent experiments). (H) Flow cytometric analysis of the expression of CTLA4 by T_reg and Foxp3 by CD4⁺CD25⁺ T cells isolated from spleen of mixed bone marrow chimeras. These data are representative results of three independent experiments (left panel). Flow cytometric analysis of the expression of ICOS by splenic T_reg cells from tam-iCD4TR2 and control mice at indicated time point p.a. Representative data of three independent experiments.</p

Development of lethal autoimmunity after thymic deletion of TR2.

<p>Tamoxifen-treatment of Rag1^−/− mice started 3 d before reconstitution with T-cell–depleted bone marrow from iCD4TR2 or control mice (A–G). (A) Flow cytometric analysis of TR2 expression by CD4⁺ and CD8⁺ T cells at day 34. Representative data of two independent experiments. (B) Body weight was monitored during the whole experiment (mean ± SEM, 5 mice per group, representative data of two independent experiments). (C) Kaplan-Meyer survival graph for all animals of experiments. (D) Representative micrographs of H&E- and anti-CD3-stained tissue sections of indicated organs at day 34. The size bar indicates 100 µm. (E) Flow cytometric analysis of the expression of CD44 and CD62l by CD4⁺ and CD8⁺ T cells. The percentage of T_n and T_em cells in the spleen of experimental and control chimeric mice (mean ± SEM, 6 mice per group, analysed in two independent experiments). (F) Percentage and number of splenic T_reg cells at day 34 (mean ± SEM, 5 mice per group; representative data of two independent experiments). (G) Flow cytometric analysis of FoxP3 and CTLA-4 by indicated splenic CD4⁺ T cells subsets at day 34 (representative data of three independent experiments). Mean florescence intensity of CTLA-4 expression by splenic T_reg cells (right panel, mean ± SEM, 4 mice per group; representative data of three independent experiments). (H) Rag1^−/− mice were reconstituted with T-cell–depleted bone marrow from iCD4TR2 or control mice. Tamoxifen treatment of recipients started 5 wk postreconstitution and body weight was monitored during the whole experiment (mean ± SEM, 5 mice per group; representative data of two independent experiments). (I) The percentage of T_em, cells in the spleen of experimental and control chimeric mice (mean ± SEM, 6 mice per group, analysed in two independent experiments). (J) Tamoxifen-treatment of Rag1^−/− mice started 3 d before reconstitution with T-cell–depleted bone marrow from iCD4TR2 or control mice. At day 15 posttransfer treatment with anti-CD8 antibody or isotype control started. Shown is a Kaplan-Meyer survival graph for all animals in the experiments (representative data of two independent experiments). (K) Representative micrographs of H&E-stained lung sections in the terminal stage of the disease. The size bar indicates 100 µm.</p

TGF-β signalling is required for CD4⁺ T cell homeostasis but dispensable for regulatory T cell function

TGF-β is widely held to be critical for the maintenance and function of regulatory T (T(reg)) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-β receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-β-driven peripheral tolerance is not regulated by TGF-β signalling on mature CD4⁺ T cells. Inducible TR2 ablation specifically on CD4⁺ T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4⁺ T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4⁺ T cells does not result in the collapse of the T(reg) cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-β signalling and the TR2-deficient T(reg) cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-β signalling on mature CD4⁺ T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice