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

Bone Morphogenic Proteins are Immunoregulatory Cytokines Controlling FOXP3+ T\u3csub\u3ereg\u3c/sub\u3e Cells

Bone morphogenic proteins (BMPs) are members of the transforming growth factor β (TGF-β) cytokine family promoting differentiation, homeostasis, and self-renewal of multiple tissues. We show that signaling through the bone morphogenic protein receptor 1α (BMPR1α) sustains expression of FOXP3 in Treg cells in peripheral lymphoid tissues. BMPR1α signaling promotes molecular circuits supporting acquisition and preservation of Treg cell phenotype and inhibiting differentiation of pro-inflammatory effector Th1/Th17 CD4+ T cell. Mechanistically, increased expression of KDM6B (JMJD3) histone demethylase, an antagonist of the polycomb repressive complex 2, underlies lineage-specific changes of T cell phenotypes associated with abrogation of BMPR1α signaling. These results reveal that BMPs are immunoregulatory cytokines mediating maturation and stability of peripheral FOXP3+ regulatory T cells (Treg cells) and controlling generation of iTreg cells. Thus, we establish that BMPs, a large cytokine family, are an essential link between stromal tissues and the adaptive immune system involved in sustaining tissue homeostasis by promoting immunological tolerance

Self and Microbiota-Derived Epitopes Induce CD4⁺ T Cell Anergy and Conversion into CD4⁺Foxp3⁺ Regulatory Cells

The physiological role of T cell anergy induction as a key mechanism supporting self-tolerance remains undefined, and natural antigens that induce anergy are largely unknown. In this report, we used TCR sequencing to show that the recruitment of CD4+CD44+Foxp3−CD73+FR4+ anergic (Tan) cells expands the CD4+Foxp3+ (Tregs) repertoire. Next, we report that blockade in peripherally-induced Tregs (pTregs) formation due to mutation in CNS1 region of Foxp3 or chronic exposure to a selecting self-peptide result in an accumulation of Tan cells. Finally, we show that microbial antigens from Akkermansia muciniphila commensal bacteria can induce anergy and drive conversion of naive CD4+CD44-Foxp3− T (Tn) cells to the Treg lineage. Overall, data presented here suggest that Tan induction helps the Treg repertoire to become optimally balanced to provide tolerance toward ubiquitous and microbiome-derived epitopes, improving host ability to avert systemic autoimmunity and intestinal inflammation

Flow cytometry, gene expression and functional analysis of effector and T_reg cells in TCR^mini-Foxp3^GFP mice bearing B16 melanoma tumors expressing NP-Ep63K.

<p>(A) Cells from control, draining lymph nodes and tumor infiltrates were isolated and single cell suspensions were stained with the relevant antibodies. Expression of Foxp3^GFP (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 Foxp3^GFP- 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 Foxp3^GFPlo (left column, broken line) and Foxp3^GFPhi (left column, dotted line) T_reg 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⁺Foxp3^GFP- (upper panels) and CD4⁺Foxp3^GFP+ (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 Foxp3^GFP- and Foxp3^GFP+ 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) Foxp3^GFP+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.</p