Indoximod-based chemo-immunotherapy for pediatric brain tumors: A first-in-children phase I trial.

BACKGROUND: Recurrent brain tumors are the leading cause of cancer death in children. Indoleamine 2,3-dioxygenase (IDO) is a targetable metabolic checkpoint that, in preclinical models, inhibits anti-tumor immunity following chemotherapy. METHODS: We conducted a phase I trial (NCT02502708) of the oral IDO-pathway inhibitor indoximod in children with recurrent brain tumors or newly diagnosed diffuse intrinsic pontine glioma (DIPG). Separate dose-finding arms were performed for indoximod in combination with oral temozolomide (200 mg/m2/day x 5 days in 28-day cycles), or with palliative conformal radiation. Blood samples were collected at baseline and monthly for single-cell RNA-sequencing with paired single-cell T cell receptor sequencing. RESULTS: Eighty-one patients were treated with indoximod-based combination therapy. Median follow-up was 52 months (range 39-77 months). Maximum tolerated dose was not reached, and the pediatric dose of indoximod was determined as 19.2 mg/kg/dose, twice daily. Median overall survival was 13.3 months (n = 68, range 0.2-62.7) for all patients with recurrent disease and 14.4 months (n = 13, range 4.7-29.7) for DIPG. The subset of n = 26 patients who showed evidence of objective response (even a partial or mixed response) had over 3-fold longer median OS (25.2 months, range 5.4-61.9, p = 0.006) compared to n = 37 nonresponders (7.3 months, range 0.2-62.7). Four patients remain free of active disease longer than 36 months. Single-cell sequencing confirmed emergence of new circulating CD8 T cell clonotypes with late effector phenotype. CONCLUSIONS: Indoximod was well tolerated and could be safely combined with chemotherapy and radiation. Encouraging preliminary evidence of efficacy supports advancing to Phase II/III trials for pediatric brain tumors.

The Simple prEservatioN of Single cElls method for cryopreservation enables the generation of single-cell immune profiles from whole blood.

Introduction: Current multistep methods utilized for preparing and cryopreserving single-cell suspensions from blood samples for single-cell RNA sequencing (scRNA-seq) are time-consuming, requiring trained personnel and special equipment, so limiting their clinical adoption. We developed a method, Simple prEservatioN of Single cElls (SENSE), for single-step cryopreservation of whole blood (WB) along with granulocyte depletion during single-cell assay, to generate high quality single-cell profiles (SCP). Methods: WB was cryopreserved using the SENSE method and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved using the traditional density-gradient method (PBMC method) from the same blood sample (n=6). The SCPs obtained from both methods were processed using a similar pipeline and quality control parameters. Further, entropy calculation, differential gene expression, and cellular communication analysis were performed to compare cell types and subtypes from both methods. Results: Highly viable (86.3 ± 1.51%) single-cell suspensions (22,353 cells) were obtained from the six WB samples cryopreserved using the SENSE method. In-depth characterization of the scRNA-seq datasets from the samples processed with the SENSE method yielded high-quality profiles of lymphoid and myeloid cell types which were in concordance with the profiles obtained with classical multistep PBMC method processed samples. Additionally, the SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. Overall, the SENSE and PBMC methods processed samples exhibited transcriptional, and cellular communication network level similarities across cell types with no batch effect except in myeloid lineage cells. Discussion: Comparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.

The Transcription Factor RXRα in CD11c+ APCs Regulates Intestinal Immune Homeostasis and Inflammation.

APCs such as dendritic cells and macrophages play a pivotal role in mediating immune tolerance and restoring intestinal immune homeostasis by limiting inflammatory responses against commensal bacteria. However, cell-intrinsic molecular regulators critical for programming intestinal APCs to a regulatory state rather than an inflammatory state are unknown. In this study, we report that the transcription factor retinoid X receptor α (RXRα) signaling in CD11c+ APCs is essential for suppressing intestinal inflammation by imparting an anti-inflammatory phenotype. Using a mouse model of ulcerative colitis, we demonstrated that targeted deletion of RXRα in CD11c+ APCs in mice resulted in the loss of T cell homeostasis with enhanced intestinal inflammation and increased histopathological severity of colonic tissue. This was due to the increased production of proinflammatory cytokines that drive Th1/Th17 responses and decreased expression of immune-regulatory factors that promote regulatory T cell differentiation in the colon. Consistent with these findings, pharmacological activation of the RXRα pathway alleviated colitis severity in mice by suppressing the expression of inflammatory cytokines and limiting Th1/Th17 cell differentiation. These findings identify an essential role for RXRα in APCs in regulating intestinal immune homeostasis and inflammation. Thus, manipulating the RXRα pathway could provide novel opportunities for enhancing regulatory responses and dampening colonic inflammation.

IRAK1-regulated IFN-γ signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies.

BACKGROUND: Stem Cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease which can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of fibroblast growth factor receptor-1 (FGFR1) as a result of chromosome translocations with poor outcome. Mouse models have been developed which faithfully recapitulate the human disease and have been used to characterize the molecular genetic events that are associated with development and progression of the disease. METHODS: CRISPR/Cas9 approaches were used to generate SCLL cells null for Interleukin receptor associated kinase 1 (IRAK1) and interferon gamma (IFNG) which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition and activity were monitored using flow cytometry. Bead-based immunoassays were used to compare the cytokine and chemokine profiles of control and knock out (KO) cells. Antibody mediated, targeted depletion of T cell and MDSCs were performed to evaluate their role in antitumor immune responses. RESULTS: In SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 KO SCLL cells were xenografted into immunocompetent syngeneic mice where the typical rapid progression of disease was lost and the mice remained disease free. IRAK1 in this system has no effect on cell cycle progression or apoptosis and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells, and tumor killing assays confirmed the role of T cells in tumor clearance. Analysis of the immune cell profile in mice transplanted with the IRAK1 expressing mock control (MC) cells shows that there is an increase in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant decrease in CD4+/CD8+ T-cell levels. MDSC suppression assays and depletion experiments showed that these MDSCs were responsible for suppression of the T cell mediated leukemia cell elimination. Immuno-profiling of a panel of secreted cytokines and chemokines showed that activation of IFN-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment with interferon gamma receptor-1 (IFNGR1) null mice and IFNG KO SCLL cells, showed the leukemia cells produced IFN-γ directly participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells. CONCLUSIONS: IRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of MDSCs via regulation of IFN-γ signaling from leukemia cells, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance.

Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

A Tryptophan-Deficient Diet Induces Gut Microbiota Dysbiosis and Increases Systemic Inflammation in Aged Mice.

The gut microflora is a vital component of the gastrointestinal (GI) system that regulates local and systemic immunity, inflammatory response, the digestive system, and overall health. Older people commonly suffer from inadequate nutrition or poor diets, which could potentially alter the gut microbiota. The essential amino acid (AA) tryptophan (TRP) is a vital diet component that plays a critical role in physiological stress responses, neuropsychiatric health, oxidative systems, inflammatory responses, and GI health. The present study investigates the relationship between varied TRP diets, the gut microbiome, and inflammatory responses in an aged mouse model. We fed aged mice either a TRP-deficient (0.1%), TRP-recommended (0.2%), or high-TRP (1.25%) diet for eight weeks and observed changes in the gut bacterial environment and the inflammatory responses via cytokine analysis (IL-1a, IL-6, IL-17A, and IL-27). The mice on the TRP-deficient diets showed changes in their bacterial abundance of Coriobacteriia class, Acetatifactor genus, Lachnospiraceae family, Enterococcus faecalis species, Clostridium sp genus, and Oscillibacter genus. Further, these mice showed significant increases in IL-6, IL-17A, and IL-1a and decreased IL-27 levels. These data suggest a direct association between dietary TRP content, the gut microbiota microenvironment, and inflammatory responses in aged mice models.

Phase II trial of the IDO pathway inhibitor indoximod plus pembrolizumab for the treatment of patients with advanced melanoma.

BACKGROUND: The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity. Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity. In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma. METHODS: Patients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma. Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I). Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label. RESULTS: Between July 2014 and July 2017, 131 patients were enrolled. (P) was used more frequently (n=114, 87%) per investigator's choice. The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%. Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9). The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients. The combination was well tolerated, and side effects were similar to what was expected from single agent (P). CONCLUSION: In this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.

Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo.

Chronic bone degenerative diseases represent a major threat to the health and well-being of the population, particularly those with advanced age. This study isolated exosomes (EXO), natural nano-particles, from dendritic cells, the "directors" of the immune response, to examine the immunobiology of DC EXO in mice, and their ability to reprogram immune cells responsible for experimental alveolar bone loss in vivo. Distinct DC EXO subtypes including immune-regulatory (regDC EXO), loaded with TGFB1 and IL10 after purification, along with immune stimulatory (stimDC EXO) and immune "null" immature (iDCs EXO) unmodified after purification, were delivered via I.V. route or locally into the soft tissues overlying the alveolar bone. Locally administrated regDC EXO showed high affinity for inflamed sites, and were taken up by both DCs and T cells in situ. RegDC EXO-encapsulated immunoregulatory cargo (TGFB1 and IL10) was protected from proteolytic degradation. Moreover, maturation of recipient DCs and induction of Th17 effectors was suppressed by regDC EXO, while T-regulatory cell recruitment was promoted, resulting in inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss. This work is the first demonstration of DC exosome-based therapy for a degenerative alveolar bone disease and provides the basis for a novel treatment strategy.

Stimulator of interferon genes agonists attenuate type I diabetes progression in NOD mice.

Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αß (IFN-αß), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αß in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy.

Cytokeratin-8 in Anaplastic Thyroid Carcinoma: More Than a Simple Structural Cytoskeletal Protein.

Anaplastic thyroid carcinoma (ATC) is almost universally fatal. Elevated keratin-8 (KRT8) protein expression is an established diagnostic cancer biomarker in several epithelial cancers (but not ATC). Several keratins, including KRT8, have been suggested to have a role in cell biology beyond that of structural cytoskeletal proteins. Here, we provide evidence that KRT8 plays a direct role in the growth of ATCs. Genomic and transcriptomic analysis of >5000 patients demonstrates that KRT8 mutation and copy number amplification are frequently evident in epithelial-derived cancers. Carcinomas arising from diverse tissues exhibit KRT8 mRNA and protein overexpression when compared to normal tissue levels. Similarly, in a panel of patient-derived ATC cell lines and patient tumors, KRT8 expression shows a similar pattern. sh-RNA-mediated KRT8 knockdown in these cell lines increases apoptosis, whereas forced overexpression of KRT8 confers resistance to apoptosis under peroxide-induced cell stress conditions. We further show that KRT8 protein binds to annexin A2, a protein known to mediate apoptosis as well as the redox pathway.

Activation of p53 in Immature Myeloid Precursor Cells Controls Differentiation into Ly6c+CD103+ Monocytic Antigen-Presenting Cells in Tumors.

CD103+ dendritic cells are critical for cross-presentation of tumor antigens. Here we have shown that during immunotherapy, large numbers of cells expressing CD103 arose in murine tumors via direct differentiation of Ly6c+ monocytic precursors. These Ly6c+CD103+ cells could derive from bone-marrow monocytic progenitors (cMoPs) or from peripheral cells present within the myeloid-derived suppressor cell (MDSC) population. Differentiation was controlled by inflammation-induced activation of the transcription factor p53, which drove upregulation of Batf3 and acquisition of the Ly6c+CD103+ phenotype. Mice with a targeted deletion of p53 in myeloid cells selectively lost the Ly6c+CD103+ population and became unable to respond to multiple forms of immunotherapy and immunogenic chemotherapy. Conversely, increasing p53 expression using a p53-agonist drug caused a sustained increase in Ly6c+CD103+ cells in tumors during immunotherapy, which markedly enhanced the efficacy and duration of response. Thus, p53-driven differentiation of Ly6c+CD103+ monocytic cells represents a potent and previously unrecognized target for immunotherapy.

Long-term sustainable dendritic cell-specific depletion murine model for periodontitis research.

Dendritic cells (DCs) are specialized antigen-presenting cells that play a pivotal role in the pathogenesis of periodontitis. The use of animal models to study the role of DCs in periodontitis has been limited by lack of a method for sustained depletion of DCs. Hence, the objectives of this study were to validate the zDC-DTR knockin mouse model of conventional DCs (cDCs) depletion, as well as to investigate whether this depletion could be sustained long enough to induce alveolar bone loss in this model. zDC-DTR mice were treated with different dose regimens of diphtheria toxin (DT) to determine survival rate. A loading DT dose of 20ng/bw, followed and maintained with doses of 10ng/bm every 3days for up to 4weeks demonstrated 80% survival. Animals were weighed weekly and peripheral blood was obtained to confirm normal neutrophil counts. Five animals per group were euthanized at baseline, 24h, 1 and 4weeks. Bone marrow (BM), spleen (SP) and gingival tissue (GT) were harvested, and cells were isolated, separated and stained for Pre-DCs precursors (CD45R-MHCII+CD11c+Flt3+CD172a+) in BM, cDCs (CD11c+MHCII+CD209+) in spleen, and DCs in GT (CD45R+MHCII+CD11c+ DC-SIGN/CD209+). Pre-DCs in BM were significantly depleted at 24h and depletion maintained for up to 4weeks, as compared to blank (PBS) controls. Circulating cDCs in spleen demonstrated a non-significant trend to deplete in 1week with high variability among mice. GT also showed a similar non-significant trend to deplete in 24h. The zDC-DTR model seems to be viable for evaluating the role of DCs immune homeostasis disruption and alveolar bone loss pathogenesis in response to long-term oral infection.

Reduction of T cell receptor diversity in NOD mice prevents development of type 1 diabetes but not Sjögren's syndrome.

Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren's syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-Ag7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRß chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRαmini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4+ T cells, as compared to the TCRmini repertoire on B6 background. Interestingly, neither transgenic TCRß nor TCRmini mice develop diabetes, which we show is due to lack of insulin B:9-23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.

Generation of T cell hybridomas from naturally occurring FoxP3+ regulatory T cells.

Generation of regulatory T cells (or Treg) derived hybridomas offers a tool to study their antigen specificity. T cells hybridomas are produced by fusing TCR α-ß-thymoma BW5147 with highly dividing T cell population. In vitro anergy of Tregs is an obstacle in generation of highly dividing Treg population for their fusion. In this chapter, we describe a simple and efficient method to generate large number of blasting Treg and their successful fusion with thymoma BW5147. The resultant hybridomas lose Treg-specific transcription factor FoxP3, respond to antigenic stimulation by producing IL-2, and thus allow the evaluation of antigen specific, Tregs-derived TCRs.

Foxp3-deficient regulatory T cells do not revert into conventional effector CD4+ T cells but constitute a unique cell subset.

Homeostasis in the immune system is maintained by specialized regulatory CD4(+) T cells (T(reg)) expressing transcription factor Foxp3. According to the current paradigm, high-affinity interactions between TCRs and class II MHC-peptide complexes in thymus "instruct" developing thymocytes to up-regulate Foxp3 and become T(reg) cells. However, the loss or down-regulation of Foxp3 does not disrupt the development of T(reg) cells but abrogates their suppressor function. In this study, we show that Foxp3-deficient T(reg) cells in scurfy mice harboring a null mutation of the Foxp3 gene retained cellular features of T(reg) cells including in vitro anergy, impaired production of inflammatory cytokines, and dependence on exogenous IL-2 for proliferation and homeostatic expansion. Foxp3-deficient T(reg) cells expressed a low level of activation markers, did not expand relative to other CD4(+) T cells, and produced IL-4 and immunomodulatory cytokines IL-10 and TGF-beta when stimulated. Global gene expression profiling revealed significant similarities between T(reg) cells expressing and lacking Foxp3. These results argue that Foxp3 deficiency alone does not convert T(reg) cells into conventional effector CD4(+) T cells but rather these cells constitute a distinct cell subset with unique features.

Tie2cre-induced inactivation of the miRNA-processing enzyme Dicer disrupts invariant NKT cell development.

MicroRNAs (miRNAs) are a class of evolutionarily conserved small noncoding RNAs that are increasingly being recognized as important regulators of gene expression. The ribonuclease III enzyme Dicer is essential for the processing of miRNAs. CD1d-restricted invariant natural killer T (iNKT) cells are potent regulators of diverse immune responses. The role of Dicer-generated miRNAs in the development and function of immune regulatory iNKT cells is unknown. Here, we generated a mouse strain with a tissue-specific disruption of Dicer, and showed that lack of miRNAs after the deletion of Dicer by Tie2-Cre (expressed in hematopoietic cells and endothelial cells) interrupted the development and maturation of iNKT cells in the thymus and significantly decreased the number of iNKT cells in different immune organs. Thymic and peripheral iNKT cell compartments were changed in miRNA-deficient mice, with a significantly increased frequency of CD4(+)CD8(+) iNKT cells in the thymus and a significantly decreased frequency of CD4(+) iNKT cells in the spleen. MiRNA-deficient iNKT cells display profound defects in alpha-GalCer-induced activation and cytokine production. Bone marrow (BM) from miRNA-deficient mice poorly reconstituted iNKT cells compared to BM from WT mice. Also, using a thymic iNKT cell transfer model, we found that iNKT cell homeostasis was impaired in miRNA-deficient recipient mice. Our data indicate that miRNAs expressed in hematopoietic cells and endothelial cells are potent regulators of iNKT cell development, function, and homeostasis.

The T-cell receptor repertoire of regulatory T cells.

The CD4(+) CD25(+) regulatory population of T cells (Treg cells), which expresses the forkhead family transcription factor (Foxp3), is the key component of the peripheral tolerance mechanism that protects us from a variety of autoimmune diseases. Experimental evidence shows that Treg cells recognize a wide range of antigenic specificities with increased reactivity to self antigens, although the affinity of these interactions remains to be further defined. The Treg repertoire is highly diverse with a distinct set of T-cell receptors (TCRs), and yet is overlapping to some extent with the repertoire of conventional T cells (Tconv cells). The majority of Treg cells are generated in the thymus. However, the role of the TCR specificity in directing thymic precursors to become Treg or Tconv cells remains unclear. On the one hand, the higher self reactivity of Treg cells and utilization of different TCRs in Treg and Tconv repertoires suggest that in TCR interactions an initial decision is made about the 'suitability' of a developing thymocyte to become a Treg cell. On the other hand, as Treg cells can recognize a wide range of foreign antigens, have a diverse TCR repertoire, and show some degree of overlap with Tconv cells, the signals through the TCR may be complementary to the TCR-independent process that generates precursors of Treg cells. In this review, we discuss how different features of the Treg repertoire influence our understanding of Treg specificities and the role of self reactivity in the generation of this population.

Nonself-antigens are the cognate specificities of Foxp3+ regulatory T cells.

The majority of regulatory Foxp3+CD4+ T cells naturally arises in the thymus. It has been proposed that T cell receptors (TCRs) on these cells recognize self-MHC class II-peptide complexes with high or higher affinity and that their specificities mirror specificities of autoreactive T cells. Here, we analyzed hundreds of TCRs derived from regulatory or nonregulatory T cells and found little evidence that the former population preferably recognizes self-antigens as agonists. Instead, these cells recognized foreign MHC-peptide complexes as often as nonregulatory T cells. Our results show that high-affinity, autoreactive TCRs are rare on all CD4+ T cells and suggest that selecting self-peptide is different from the peptide that activates the same regulatory T cells in the periphery.

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells.

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.

Presentation of antagonist peptides to naive CD4+ T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells.

By using dendritic cells (DCs) transduced with retroviruses encoding covalent A(b)beta/peptide fusion proteins tagged with fluorescent proteins, we followed the relocation of class II MHC molecules loaded with agonist or null peptides during the onset of activation of naive and effector CD4(+) T cells. Clusters of T cell receptor (TCR)/CD3 complex formed in parallel with clusters of agonist class II MHC/peptide complexes on the surface of DCs. However, activation of naive but not effector T cells was accompanied by expulsion of the null class II MHC/peptide complexes from the T cell-DC interface. These effects were perturbed in the presence of exogenously supplied antagonist peptide. These results suggest that interference with selective relocation of agonist and null MHC/peptide complexes in the immunological synapse contributes to the inhibitory effect of antagonist peptides on the response of naive CD4(+) T cells to agonist ligands.