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

Dormant Pathogenic CD4(+) T Cells Are Prevalent in the Peripheral Repertoire of Healthy Mice

Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4+Foxp3− T cells escape negative selection and in the periphery require continuous suppression by CD4+Foxp3+ regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4+Foxp3− cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including selfpeptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4+ T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage

Analysis of the TCR repertoires of naive, activated and T_reg cells in control and draining lymph nodes and tumor lesions of tumor-bearing TCR^mini-Foxp3^GFP mice.

<p>(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 T_reg 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.</p

Analysis of the TCR repertoire in tumor-bearing TCR^mini-Foxp3^GFP mice.

<p>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 TCR^mini-Foxp3^GFP 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 TCR^mini-Foxp3^GFP mice with tumors. 20 most abundant clones in the control lymph nodes is shown. (C) Analysis of frequency of activated (Activ.) and T_reg 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 T_reg subset (R) in the populations of activated (blue, upper part of the panel) and T_reg (purple, lower part of the panel) cells in tumors. Clones selected for analysis are the 20 most abundant clones in the population of T_reg 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 T_reg clones (blue) expressing the exclusive set of TCRs in tumors. Clones selected for analysis are the 20 most abundant clones in the population of T_reg cells in healthy mice (purple).</p

Amino-acid sequences of TCRα chain CDR3 regions of Ep63K-specific CD4⁺ T cell hybridomas obtained from TCR^mini-Foxp3^GFP mice and generation of B16 tumors expressing tumor associated neo-antigen NP-Ep63K.

<p>(A) Amino-acid sequences of TCRα chain CDR3 regions of Ep63K-specific CD4⁺ T cell hybridomas obtained from TCR^mini-Foxp3^GFP 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 A^b are shown as clear rectangles and Ep63K peptide is shown as horizontal lines. Numbers show the range of amino acids constituting peptides binding to A^b. (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.</p