Mechanisms of CD8+ T cell-mediated tolerance and immunity in the intestinal mucosa

Das intestinale Immunsystem stellt den größten und komplexesten Teil des menschlichen Immunsystems dar und benötigt durch die exponierte Lage ein besonderes Gleichgewicht zwischen Immunität und Toleranz. Bis heute ist nicht vollständig geklärt in welchen intestinalen Kompartimenten die Induktion von T-Zell-vermittelter Immunität und Toleranz stattfindet. Zusätzlich werden immer neue T-Zell-Subpopulationen im intestinalen Immunsystem identifiziert, die phänotypisch und funktionell noch nicht charakterisiert sind. Insbesondere die Induktion von CD8+ T-Zell-Antworten in der intestinalen Mukosa ist wenig untersucht. Die Ergebnisse im ersten Teil dieser Arbeit zeigen, dass neben den mesenterischen Lymphknoten weitere lymphoide Strukturen wie die Peyer’schen Platten für die Induktion von CD8+ Effektor T-Zellantworten in der intestinalen Mukosa entscheidend sind. Trotz der operativen Resektion der mesenterischen Lymphknoten kommt es in VILLIN-HA transgenen Tieren zur Aktivierung von Darm-spezifischen CD8+ T-Zellen und einer fulminanten intestinalen Entzündung. In FTY720 Experimenten, bei denen die Migration von Lymphozyten aus lymphoiden Organen inhibiert ist, konnte die Aktivierung von CD8+ T-Zellen in den Peyer‘schen Platten nachgewiesen werden. Dabei werden intestinale Antigene von spezialisierten Dendritischen Zellen aufgenommen und an den Ort der Antigenpräsentation transportiert. Die Milz ist für das intestinale Immunsystem eher redundant, da es in der Milz im VILLIN-HA transgenen Mausmodell nicht zur Präsentation des intestinalen Antigens kommt. Im zweiten Teil dieser Arbeit wurden die Induktion und der Phänotyp von intestinalen CD8+Foxp3+ T-Zellen analysiert. Untersuchungen im VILLIN-HA transgenen Mausmodell zeigen, dass Darm-spezifische CD8+Foxp3+ T-Zellen in den Strukturen des GALT induziert werden. In in vitro Studien wurde gezeigt, dass die Induktion muriner und humaner CD8+Foxp3+ T-Zellen durch Darm-relevante Mediatoren wie TGF- und Retinsäure vermittelt wird. Umfangreiche Transkriptomanalysen dieser in vitro-induzierten CD8+Foxp3+ T-Zellen zeigen die erhöhte Expression der Treg-spezifischen Markermoleküle CD25, Gpr83 und CTLA-4. In vitro und in vivo Analysen zeigen das hohe immunsuppressive Potential dieser Zellen. Die phänotypische Untersuchung der CD8+ T-Zellpopulation im peripheren Blut von Patienten mit chronisch-entzündlicher Darmerkrankung zeigt eine deutliche Reduktion CD8+Foxp3+ T-Zellen während des akuten Schubes. Die Modulation der Anzahl CD8+Foxp3+ T-Zellen könnte daher ein neuer Ansatzpunkt zur Immuntherapie von chronisch-entzündlichen Darmerkrankungen sein

Local Induction of Immunosuppressive CD8+ T Cells in the Gut-Associated Lymphoid Tissues

Background: In contrast to intestinal CD4 + regulatory T cells (Tregs), the generation and function of immunomodulatory intestinal CD8 + T cells is less well defined. To dissect the immunologic mechanisms of CD8 + T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cellreceptor specific for HA was studied. Methodology and Principal Findings: HA-specific CD8 + T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3 + and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8 + Foxp3 + T cells. Antigen-experienced CD8 + T cells in this transgenic mouse model suppressed the proliferation of CD8 + and CD4 + T cells in vitro. Gene expression analysis of suppressive HA-specific CD8 + T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4 + T reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8 + Foxp3 + T cells. Conclusion and Significance: We demonstrate that gut specific antigen presentation is sufficient to induce CD8 + T regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells

Differentially gene expression of T_reg-associated genes by HA-specific CD8⁺ T cells isolated from MLN of CL4-TCR and VILLIN-HA/CL4-TCR transgenic mice.

<p>Differentially gene expression of T_reg-associated genes by HA-specific CD8⁺ T cells isolated from MLN of CL4-TCR and VILLIN-HA/CL4-TCR transgenic mice.</p

Specific expression of CCL4 by HA-specific CD8⁺ T cells from VILLIN-HA/CL4-TCR transgenic mice.

<p>(A) CD8⁺H-2K^dIYSTVASSL⁺ were sorted from the MLN of CL4-TCR and VILLIN-HA/CL4-TCR transgenic mice. Affymetrix gene chip experiments were performed and analyzed for the expression of CCL4. Expression of CCL4 is indicated as signal intensity. (B) CD8⁺ H-2K^dIYSTVASSL⁺ T cells were isolated from the MLN of CL4-TCR and VILLIN-HA/CL4-TCR transgenic mice by cell sorting. CCL4 expression was assayed by quantitative PCR and normalized relative to expression of RPS9. Data shown are representative of three independent experiments. (C) HA-specific CD4⁺ T cells (TCR-HA) and HA-specific CD8⁺ T cells (CL4-TCR) were stimulated with the corresponding antigen in the presence of indicated concentrations CCL4 or CCL3. Proliferation was measured by [³H]thymidine incorporation. One representative experiment out of three independent experiments is shown.</p

Peripheral induction of CD8⁺Foxp3⁺ T cells in vivo and in vitro.

<p>(A) CFSE-labeled HA-specific CD8⁺CD25⁻ T cells from CL4-TCR transgenic mice were adoptively transferred into VILLIN-HA and BALB/c recipient mice. At day 4 after adoptive transfer cells from the MLN of recipient mice were isolated and stained for the expression of CD8 and Foxp3. Histograms show proliferation of HA-specific CD8⁺ T cells by loss of CFSE dye, dot plot demonstrates the expression of Foxp3 in proliferating CD8⁺ T cells. (B) DCs from MLN of VILLIN-HA and BALB/c mice were co-cultured with CFSE-labeled HA-specific CD8⁺CD25⁻ T cells from CL4-TCR transgenic mice for 5 days. Cells were stained for the expression of CD8 and Foxp3. Histograms show proliferation of HA-specific CD8⁺ T cells by loss of CFSE-dye, dot plot demonstrates the expression of Foxp3 in proliferating CD8⁺ T cells. (C) CFSE-labeled HA-specific CD8⁺CD25⁻ T cells from CL4-TCR transgenic mice were co-cultured with MLN DCs from BALB/c mice and exogenous HA peptide for 4 days. Where indicated, cells were supplemented with 2 ng/ml human rTGF-β or 100 nM RA. Cells were stained for the expression of CD8 and Foxp3. Gating on proliferating CD8⁺ T cells, the expression of Foxp3 vs CFSE fluorescence intensity is demonstrated. Data shown are representative of three independent experiments. (D) For in vitro T cell suppression assays, HA-specific CD8⁺ T cells were separated into CD8⁺Foxp3⁻/GFP^- and CD8⁺Foxp3⁺/GFP⁺ T cells by FACS on CD8 and GFP expression. Sorted T cells were co-cultured with freshly isolated CFSE-labeled HA-specific CD4⁺CD25⁻ responder T cells and APCs, and stimulated with the cognate HA peptides. Histograms show proliferation of responder T cells as determined by loss of CFSE dye. Data from two independent experiments are shown.</p

Phenotype of HA-specific CD8⁺ T cells from VILLIN-HA/CL4-TCR transgenic mice.

<p>(A) Cells isolated from the MLN of CL4-TCR and VILLIN-HA/CL4-TCR transgenic mice were stained for the expression of CD8, H-2K^d IYSTVASSL, CD103 and Nrp1. Dot plots represent the percentage of HA-specific CD8⁺ T cells expressing the indicated molecules. (B) HA-specific CD8⁺ T cells were isolated from the MLN of CL4-TCR and VILLIN-HA/CL4-TCR transgenic mice by cell sorting. CD83 expression was assayed by quantitative RT-PCR and normalized relative to expression of RPS9. Data shown are representative of three independent experiments.</p

Induction of antigen-specific CD8⁺Foxp3⁺ T cells in vivo.

<p>Lymphocytes from the thymus (TH), spleen (SP) and mesenteric lymph nodes (MLN) of CL4-TCR, VILLIN-HA/CL4-TCR and VILLIN-HA transgenic mice were stained for the expression of CD8 and H-2K^dIYSTVASSL and analyzed regarding the expression of Foxp3. Percentage of Foxp3⁺ T cells is indicated. One representative experiment out of four independent experiments with similar results is shown.</p

Transient ablation of regulatory T cells improves antitumor immunity in colitis-associated colon cancer

Abstract Regulatory T cells (Treg) are supportive to cancer development in most tissues, but their role in colitis-associated colon cancer (CAC) remains unclear. In this study, we investigated the role of CD4+Foxp3+ Treg in a mouse model of CAC and in patients with colon cancer. These Treg were increased strongly in number in a mouse model of CAC and in the peripheral blood of patients with colon cancer, exhibiting an activated phenotype as defined by elevated expression of GARP, CD103, CTLA-4, and IL10, along with an increased suppressive effect on the proliferation and Th1 cytokine expression of CD4+CD25− responder T cells ex vivo. Transient ablation of CD4+Foxp3+ Treg during tumor development in the CAC model suppressed tumor outgrowth and distribution, accompanied by an increased number of CD8+IFNγ/granzyme B-producing effector T cells. Conversely, inactivation of IL10 in Treg did not elevate the antitumor response but instead further boosted tumor development. Our results establish a tumor-promoting function for Treg during CAC formation, but they also suggest that a selective, transient ablation of Treg can evoke antitumor responses, with implications for immunotherapeutic interventions in patients with CAC. Cancer Res; 74(16); 4258–69. ©2014 AACR.</jats:p

Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes

Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3(+) regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance