Figure 1.
Amino-acid sequences of TCRα chain CDR3 regions of Ep63K-specific CD4+ T cell hybridomas obtained from TCRmini-Foxp3GFP mice and generation of B16 tumors expressing tumor associated neo-antigen NP-Ep63K.
(A) Amino-acid sequences of TCRα chain CDR3 regions of Ep63K-specific CD4+ T cell hybridomas obtained from TCRmini-Foxp3GFP mice. (B) Production of the nucleoprotein-Ep63K-GFP expression construct. Sections encoding nucleoprotein are shown as dotted rectangles and GFP is shown as vertical lines. Parts of the nucleoprotein encoding peptides binding Ab are shown as clear rectangles and Ep63K peptide is shown as horizontal lines. Numbers show the range of amino acids constituting peptides binding to Ab. (C) Flow cytometry analysis of the B16 melanoma cells expressing nucleoprotein-Ep63K-GFP. B16 melanoma cells were transduced with the LZRS-pBMN-Z retroviral vector expressing nucleoprotein-Ep63K-GFP fusion protein (lower histogram) or a control vector (upper histogram). (D) Recombinant nucleoprotein-Ep63K is processed by bone marrow derived dendritic cells and Ep63K peptide is recognized by specific CD4+ hybridoma 123.3. IL-2 (pg/ml) production in the supernatant of dendritic cells cultured without and with 0.9 or 1.8 µg/ml of the recombinant protein.
Figure 2.
Flow cytometry, gene expression and functional analysis of effector and Treg cells in TCRmini-Foxp3GFP mice bearing B16 melanoma tumors expressing NP-Ep63K.
(A) Cells from control, draining lymph nodes and tumor infiltrates were isolated and single cell suspensions were stained with the relevant antibodies. Expression of Foxp3GFP (left column) and CD25 (second column) on CD4+ T cells are shown. Expression of activation markers CD44 and CD62L is shown on gated effector CD4+ Foxp3− cells (third column) and Foxp3 and CD25 expression are shown on gated CD4+ T cells (right column). Gates used to define Foxp3GFP- cells (left column, continuous line) and subsets of naive CD44−CD62L+ (third column, continuous line) and activated CD44+CD62L− (third column, dotted line) cells as well as Foxp3GFPlo (left column, broken line) and Foxp3GFPhi (left column, dotted line) Treg cells for TCR repertoire studies are shown as rectangles. Naive cells were absent in tumors. Figure shows representative data of at least five mice analyzed. (B) Expression of GITR (left panels) and CTLA-4 (right panels) in CD4+Foxp3GFP- (upper panels) and CD4+Foxp3GFP+ (lower panels) cells isolated from tumor draining lymph nodes and tumors. Two individual mice were analyzed. (C) Analysis of Foxp3, IL-10 and TGF-β expression in Foxp3GFP- and Foxp3GFP+ CD4+ T cells isolated from tumors. Sorted cells were lysed directly and gene expression was detected by RT-PCR. Samples were normalized for β-actin expression. Two individual mice were analyzed. (D) Foxp3GFP+CD4+ T cells isolated from the draining lymph nodes (DrLN) or tumors (TILs) suppress proliferation of effector CD4+ T cells. One of two experiments is shown.
Figure 3.
Analysis of TCR repertoires using 2D-F-SSCP.
(A) Spot patterns representing TCR repertoires of naive, activated effector CD4+ T cells and Foxp3GFPlo and Foxp3GFPhi Treg cells isolated from control and draining lymph nodes and tumors of tumor-bearing TCRmini-Foxp3GFP mice. Blue asterisks indicate control spots used to overlay gel images for analysis. Spots representing T cells expressing the same TCR as Ep63K-specific hybridoma #274 are shown in the populations of Foxp3GFPlo and Foxp3GFPhi cells in tumors. The number of spots defined on each gel by image analysis software is shown in lower left corner of each gel image. Similarity between gels is shown as percentage of overlapping spots. Gels compared are indicated by arrows. (B) Pair wise comparison of the total number and number of overlapping spots between naive, activated and Treg CD4+ T cell populations isolated from control and draining lymph nodes and tumors of tumor-bearing TCRmini-Foxp3GFP mice. Left table includes comparison of cell populations isolated from different anatomical locations, right upper table shows comparisons of cell populations isolated from the same organ and right lower table shows comparisons of different cell populations isolated from different organs.
Figure 4.
Analysis of the TCR repertoires of naive, activated and Treg cells in control and draining lymph nodes and tumor lesions of tumor-bearing TCRmini-Foxp3GFP mice.
(A) Estimation of the TCR repertoire diversity using Chao mean. The data range spanned by vertical lines represent 95% confidence interval for Chao mean. The circles represent values of Chao estimator. (B) Estimation of the similarities of the TCR repertoires presented as a dendrogram based on the differences of the relative entropy against the pooled population for TCRs expressed by naive, activated (Activ.) and Treg cells isolated from control (Ctrl. LN) and draining (Dr. LN) lymph nodes and tumor infiltrate (TILs). The dendrogram construction begins with each cell subset being a separate cluster. Then, the most similar cell populations (with the smallest difference in their relative entropies) are joined. We continue the process until we obtain a single cluster. The distance between two clusters is taken as the maximum difference in relative entropies of their members.
Figure 5.
Analysis of the TCR repertoire in tumor-bearing TCRmini-Foxp3GFP mice.
Sequences of the TCRα chain CDR3 regions are shown below plots, red sequences mark T cell clones specific for Ep63K peptide. Numbers indicate the percentage of clones shown on the plot (black) and the percentage of the Ep63K-specific cells (red) in the total population of the T cell subset analyzed. (A) Naive CD4+ T cells in healthy and tumor-bearing TCRmini-Foxp3GFP mice express similar TCR repertoires. Frequencies (%) of the 20 most abundant clones in control lymph nodes of mice with tumors (dark purple) and in lymph nodes of healthy mice (light purple) are shown. (B) Comparison of TCR repertoires of naive CD4+ T cells in the control (dark purple) and the draining lymph nodes (light purple) of TCRmini-Foxp3GFP mice with tumors. 20 most abundant clones in the control lymph nodes is shown. (C) Analysis of frequency of activated (Activ.) and Treg cell clones in control (upper panel) and draining (middle panel) lymph nodes and tumors (lower panel). 20 most abundant clones in the population of activated T cells in tumors was selected for analysis. TCRs marked with “x” were also found in B16 tumors not expressing Ep63K epitope. (D) The abundance of T cell clones expressing TCRs shared with naive/effector CD4+ T cells (N) or expressing TCRs exclusive for the Treg subset (R) in the populations of activated (blue, upper part of the panel) and Treg (purple, lower part of the panel) cells in tumors. Clones selected for analysis are the 20 most abundant clones in the population of Treg cells in tumors. Receptors found in naive/effector T cells (N) and Treg cells (R) are shown above the plot. Receptors not assigned to any subset are labeled “?”. (E) The frequency of Treg clones (blue) expressing the exclusive set of TCRs in tumors. Clones selected for analysis are the 20 most abundant clones in the population of Treg cells in healthy mice (purple).
Figure 6.
Clonal abundance (%) of Ep63K-specific T cell clones in naive (sparse dots), activated (dense dots) and Treg (stripes) subsets in the control and draining lymph nodes and tumors of tumor-bearing TCRmini-Foxp3GFP mice.
The percentage of all Ep63K-specific clones in populations of naive, activated and Treg cells is shown on each plot. Names of hybridomas expressing a particular TCRα chain are shown above upper panel. Asterisks indicate that no clones were found in the indicated cell subsets.
Figure 7.
Adaptive Treg cells expressing wild-type TCR repertoire dominate Treg cell population in melanoma tumors.
TCRmini mice were inoculated with B16 melanoma tumors and, after 3 days, reconstituted with CD4+Foxp3GFP- from Ly5.1+/−Foxp3GFP mice or CD4+Foxp3GFPhi cells from Ly5.1+/+Foxp3GFP mice. Lymph nodes (A) and tumor infiltrating lymphocytes (B) were analyzed by flow cytometry 12 days after cell transfer. (A) Flow cytometry analysis of the CD4+ T cell population in tumor draining lymph nodes. Upper left dot plot shows Foxp3GFP expression (dotted line rectangle) in CD4+ T cells (dashed line rectangle). Numbers represent proportions of cells in each quadrant. Lower left dot plot shows proportions of recipient Ly5.1−/− T cells and donor Ly5.1+/+Foxp3GFPhi and Ly5.1+/−Foxp3GFP- cells in the total population of gated CD4+ T cells. Gate used to define CD4+ T cells is shown by dashed rectangle on the upper left plot. Proportions of cells expressing Foxp3GFP in donor CD4+Ly5.1+/+ and CD4+Ly5.1+/− cells are shown on upper and lower histograms respectively. Upper right dot plot shows proportions of lymph node CD4+Foxp3GFP+ T cells generated by donor Ly5.1+/+Foxp3GFPhi and Ly5.1+/−Foxp3GFP- cells. Gate used to define CD4+Foxp3GFP+ T cells is shown by dotted rectangle on the upper left dot plot. The data shown is representative of three mice analyzed. The summary of the data for gated total CD4+ cells and CD4+Foxp3GFP+ cells is presented on the bar graph. (B) Flow cytometry analysis of the CD4+ T cell population in tumors. Lymphoid cells were purified on Lympholyte-M gradient. All dot plots and histograms show the same cell subsets as analyzed in the lymph nodes.
Figure 8.
Flow cytometry analysis of CD4+ T cells stimulated with dendritic cells presenting Ep63K peptide.
Expression of Foxp3GFP and CD25 is shown on gated CD4+ T cells. (A) Analysis of the CD4+ T cells from control (left) and draining (middle) lymph nodes of TCRmini-Foxp3GFP mice immunized with dendritic cells presenting Ep63K and CD4+ T cells isolated from TCRmini-Foxp3GFP mice and stimulated with dendritic cells in vitro (right). Dendritic cells (2×105) were injected into footpads of TCRmini-Foxp3GFP mice for three days and mice were sacrificed after 5 days and popliteal lymph nodes were analyzed. (B) Analysis of gated CD4+ T cells isolated from tumors of tumor-bearing TCRmini-Foxp3GFP control mice (left) and mice immunized with dendritic cells (middle). TCRmini-Foxp3GFP mice were inoculated with B16 melanoma cells expressing NP-EP63K and at the same time dendritic cells were injected (5×104). Injections of dendritic cells continued daily until mice were sacrificed. The abundance (%) of CD4+ T cells subsets CD25-FoxpGFP+, CD25+FoxpGFP+ and CD25+FoxpGFP- in the tumor tissue of TCRmini-Foxp3GFP control (dots) and immunized (stripes) mice. At least three mice were analyzed in each group.