How Numbers, Nature, and Immune Status of Foxp3+ Regulatory T-Cells Shape the Early Immunological Events in Tumor Development

The influence of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) on cancer progression has been demonstrated in a large number of preclinical models and confirmed in several types of malignancies. Neoplastic processes trigger an increase of Treg numbers in draining lymph nodes, spleen, blood, and tumors, leading to the suppression of anti-tumor responses. Treg-depletion before or early in tumor development may lead to complete tumor eradication and extends survival of mice and humans. However this strategy is ineffective in established tumors, highlighting the critical role of the early Treg-tumor encounters. In this review, after discussing old and new concepts of immunological tumor tolerance, we focus on the nature (thymus-derived vs. peripherally derived) and status (naïve or activated/memory) of the regulatory T-cells at tumor emergence. The recent discoveries in this field suggest that the activation status of Tregs and effector T-cells (Teffs) at the first encounter with the tumor are essential to shape the fate and speed of the immune response across a variety of tumor models. The relative timing of activation/recruitment of anti-tumor cells vs. tolerogenic cells at tumor emergence appears to be crucial in the identification of tumor cells as friend or foe, which has broad implications for the design of cancer immunotherapies

Dll4-Notch signaling in Flt3-independent dendritic cell development and autoimmunity in mice

Delta-like ligand 4 (Dll4)-Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4-Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4 +CD25 +FoxP3 + regulatory T cells (T reg cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural T reg cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4 -CD8 -c-kit +CD44 +CD25 - (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4 + T cells into T reg cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of T reg cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a T reg cell-mediated mechanism and inhibits CD8+ T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased T reg cell numbers in the pancreasdraining lymph nodes. These results identify Dll4-Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated T reg cell homeostasis and autoimmunity

Flt3 signaling-dependent dendritic cells protect against atherosclerosis

Early events in atherosclerosis occur in the aortic intima and involve monocytes that become macrophages. We looked for these cells in the steady state adult mouse aorta, and surprisingly, we found a dominance of dendritic cells (DCs) in the intima. In contrast to aortic adventitial macrophages, CD11c +MHC II hi DCs were poorly phagocytic but were immune stimulatory. DCs were of two types primarily: classical Flt3-Flt3L signaling-dependent, CD103 +CD11b - DCs and macrophage-colony stimulating factor (M-CSF)-dependent, CD14 +CD11b +DC-SIGN + monocyte-derived DCs. Both types expanded during atherosclerosis. By crossing Flt3 -/- to Ldlr -/- atherosclerosis-prone mice, we developed a selective and marked deficiency of classical CD103 + aortic DCs, and they were associated with exacerbated atherosclerosis without alterations in blood lipids. Concomitantly, the Flt3 -/-Ldlr -/- mice had fewer Foxp3 + Treg cells and increased inflammatory cytokine mRNAs in the aorta. Therefore, functional DCs are dominant in normal aortic intima and, in contrast to macrophages, CD103 + classical DCs are associated with atherosclerosis protection

Continuous Activation of Autoreactive CD4+ CD25+ Regulatory T Cells in the Steady State

Despite a growing interest in CD4+ CD25+ regulatory T cells (Treg) that play a major role in self-tolerance and immunoregulation, fundamental parameters of the biology and homeostasis of these cells are poorly known. Here, we show that this population is composed of two Treg subsets that have distinct phenotypes and homeostasis in normal unmanipulated mice. In the steady state, some Treg remain quiescent and have a long lifespan, in the order of months, whereas the other Treg are dividing extensively and express multiple activation markers. After adoptive transfer, tissue-specific Treg rapidly divide and expand preferentially in lymph nodes draining their target self-antigens. These results reveal the existence of a cycling Treg subset composed of autoreactive Treg that are continuously activated by tissue self-antigens

Self-specific memory regulatory T cells protect embryos at implantation in mice

Regulatory T cells (Tregs) play crucial roles in both fetal and tumor development. We recently showed that immunosurveillance by pre-existing CD44CD62L activated/memory Tregs (amTregs) specific for self-Ags protects emergent tumor cells in mice. This Treg response of a memory type is more rapid than and dominates the antitumor response of tumor-specific effector T cells. In this study, we report striking similarities between the early Treg responses to embryo and tumor implantation. Tregs are rapidly recruited to uterus-draining lymph nodes and activated in the first days after embryo implantation in both syngeneic and allogeneic matings; express the markers of the amTreg subset; and are at least in part self-Ag specific, as seen in tumor emergence. Unlike in the tumor emergence setting, however, for which preimmunization against tumor Ags is sufficient for complete tumor eradication even in the presence of Tregs, Treg depletion is additionally required for high frequencies of fetus loss after preimmunization against paternal tissue Ags. Thus, amTregs play a major role in protecting embryos in both naive and preimmune settings. This role and the ensuing therapeutic potential are further highlighted by showing that Treg stimulation, directly by lowdose IL-2 or indirectly by Fms-related tyrosine kinase 3 ligand, led to normal pregnancy rates in a spontaneous abortion-prone model

Synthetic Melanin Acts as Efficient Peptide Carrier in Cancer Vaccine Strategy

We previously reported that a novel peptide vaccine platform, based on synthetic melanin nanoaggregates, triggers strong cytotoxic immune responses and significantly suppresses tumor growth in mice. However, the mechanisms underlying such an efficacy remained poorly described. Herein, we investigated the role of dendritic cells (DCs) in presenting the antigen embedded in the vaccine formulation, as well as the potential stimulatory effect of melanin upon these cells, in vitro by coculture experiments and ELISA/flow cytometry analysis. The vaccine efficiency was evaluated in FLT3-L−/− mice constitutively deficient in DC1, DC2, and pDCs, in Zbtb46DTR chimera mice deficient in DC1 and DC2, and in LangerinDTR mice deficient in dermal DC1 and Langerhans cells. We concluded that DCs, and especially migratory conventional type 1 dendritic cells, seem crucial for mounting the immune response after melanin-based vaccination. We also assessed the protective effect of L-DOPA melanin on peptides from enzymatic digestion, as well as the biodistribution of melanin–peptide nanoaggregates, after subcutaneous injection using [18F]MEL050 PET imaging in mice. L-DOPA melanin proved to act as an efficient carrier for peptides by fully protecting them from enzymatic degradation. L-DOPA melanin did not display any direct stimulatory effects on dendritic cells in vitro. Using PET imaging, we detected melanin–peptide nanoaggregates up to three weeks after subcutaneous injections within the secondary lymphoid tissues, which could explain the sustained immune response observed (up to 4 months) with this vaccine technology

Tumor emergence is sensed by self-specific CD44hi memory Tregs that create a dominant tolerogenic environment for tumors in mice

Early responses of Tregs and effector T cells (Teffs) to their first encounter with tumor cells have been poorly characterized. Here we have shown, in both implanted and in situ–induced mouse tumor models, that the appearance of tumor cells is immediately sensed by CD44hi memory Tregs that are specific for self antigens. The rapid response of these Tregs preceded and prevented activation of naive antitumor Teffs. The relative speed of the Treg versus the Teff response within the first 2–4 days determined the outcome of the antitumor immune response: tolerance or rejection. If antitumor memory Teffs were present at the time of tumor emergence, both Tregs and Teffs were recruited and activated with memory kinetics; however, the Tregs were unable to control the Teffs, which eradicated the tumor cells. This balance between effector and regulatory responses did not depend on the number of Tregs and Teffs, but rather on their memory status. Thus, in the natural setting, dominant tolerogenic immunosurveillance by self-specific memory Tregs protects tumors, just as it protects normal tissues. More generally, our results reveal that the timing of Treg and Teff engagement, determined by their memory status, is an important mode of regulation of immune responses

Les cellules dendritiques FLT3L-dépendantes contrôlent l'immunité tumorale en modulant l'homéostasie des lymphocytes T régulateurs et des lymphocytes Natural Killers

International audienceFLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector T cells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.Lors des cancers, les cellules dendritiques classiques (cDCs) dépendantes du FLT3-L recrutent des cellules anti-tumorales mais également des cellules protectrices des tumeurs. Nous avons évalué la croissance tumorale chez des souris présentant des quantités de cDCs faibles, normales ou élevées. Paradoxalement, le déficit comme l’excès de cDCs améliorent la survie chez les souris atteintes de mélanome. Dans le contexte de déficit en cDCs, les tumeurs sont réprimées par le système immunitaire adaptatif via un afflux de lymphocytes T effecteurs et une diminution des lymphocytes T régulateurs (Tregs) et tueurs naturels (NK). Un nombre élevé de cDCs favorise au contraire la réponse anti-tumorale innée, avec un recrutement massif de cellules NK activées, malgré une infiltration importante de Tregs. L’immunothérapie anti-CTLA-4 mais pas anti-PD-1 agit en synergie avec la thérapie FLT3-L dans le contexte élevé en cDC mais pas dans le contexte de déficit en cDC. La combinaison d’un traitement stimulateur des cDCs et de déplétion des Tregs améliore considérablement la survie des souris porteuses de tumeurs. Les données transcriptomiques confirment l'effet paradoxal des niveaux de cDCs sur la survie des patients dans de nombreux types de cancer humains. L'excès de cDC et le déficit en Treg est prédictif d’une meilleure survie des patients. Ces travaux suggèrent que la modulation des quantités de cDCs via la signalisation FLT3 a un fort potentiel thérapeutique en cancérologie