Increased CD8+ tissue resident memory T cells, regulatory T cells and activated natural killer cells in systemic sclerosis lungs.

OBJECTIVE: Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. METHODS: Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions. RESULTS: Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. CONCLUSIONS: SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.

The mycosis fungoides cutaneous microenvironment shapes dysfunctional cell trafficking, antitumor immunity, matrix interactions, and angiogenesis.

Malignant T lymphocyte proliferation in mycosis fungoides (MF) is largely restricted to the skin, implying that malignant cells are dependent on their specific cutaneous tumor microenvironment (TME), including interactions with non-malignant immune and stromal cells, cytokines, and other immunomodulatory factors. To explore these interactions, we performed a comprehensive transcriptome analysis of the TME in advanced-stage MF skin tumors by single-cell RNA sequencing. Our analysis identified cell-type compositions, cellular functions, and cell-to-cell interactions in the MF TME that were distinct from those from healthy skin and benign dermatoses. While patterns of gene expression were common among patient samples, high transcriptional diversity was also observed in immune and stromal cells, with dynamic interactions and crosstalk between these cells and malignant T lymphocytes. This heterogeneity mapped to processes such as cell trafficking, matrix interactions, angiogenesis, immune functions, and metabolism that affect cancer cell growth, migration, and invasion, as well as antitumor immunity. By comprehensively characterizing the transcriptomes of immune and stromal cells within the cutaneous microenvironment of individual MF tumors, we have identified patterns of dysfunction common to all tumors that represent a resource for identifying candidates with therapeutic potential as well as patient-specific heterogeneity that has important implications for personalized disease management.

Cell Type-Specific Biomarkers of Systemic Sclerosis Disease Severity Capture Cell-Intrinsic and Cell-Extrinsic Circuits.

OBJECTIVE: Systemic sclerosis (SSc) is a multifactorial autoimmune fibrotic disorder involving complex rewiring of cell-intrinsic and cell-extrinsic signaling coexpression networks involving a range of cell types. However, the rewired circuits as well as corresponding cell-cell interactions remain poorly understood. To address this, we used a predictive machine learning framework to analyze single-cell RNA-sequencing data from 24 SSc patients across the severity spectrum as quantified by the modified Rodnan skin score (MRSS). METHODS: We used a least absolute shrinkage and selection operator (LASSO)-based predictive machine learning approach on the single-cell RNA-sequencing data set to identify predictive biomarkers of SSc severity, both across and within cell types. The use of L1 regularization helps prevent overfitting on high-dimensional data. Correlation network analyses were coupled to the LASSO model to identify cell-intrinsic and cell-extrinsic co-correlates of the identified biomarkers of SSc severity. RESULTS: We found that the uncovered cell type-specific predictive biomarkers of MRSS included previously implicated genes in fibroblast and myeloid cell subsets (e.g., SFPR2+ fibroblasts and monocytes), as well as novel gene biomarkers of MRSS, especially in keratinocytes. Correlation network analyses revealed novel cross-talk between immune pathways and implicated keratinocytes in addition to fibroblast and myeloid cells as key cell types involved in SSc pathogenesis. We then validated the uncovered association of key gene expression and protein markers in keratinocytes, KRT6A and S100A8, with SSc skin disease severity. CONCLUSION: Our global systems analyses reveal previously uncharacterized cell-intrinsic and cell-extrinsic signaling coexpression networks underlying SSc severity that involve keratinocytes, myeloid cells, and fibroblasts.

Single-Cell RNA Sequencing Unveils the Clonal and Transcriptional Landscape of Cutaneous T-Cell Lymphomas.

PURPOSE: Clonal malignant T lymphocytes constitute only a fraction of T cells in mycosis fungoides skin tumors and in the leukemic blood of Sézary syndrome, the classic types of cutaneous T-cell lymphomas. However, lack of markers specific for malignant lymphocytes prevents distinguishing them from benign T cells, thus delaying diagnosis and the development of targeted treatments. Here we applied single-cell methods to assess the transcriptional profiles of both malignant T-cell clones and reactive T lymphocytes directly in mycosis fungoides/Sézary syndrome patient samples. EXPERIMENTAL DESIGN: Single-cell RNA sequencing was used to profile the T-cell immune repertoire simultaneously with gene expression in CD3+ lymphocytes from mycosis fungoides and healthy skin biopsies as well as from Sézary syndrome and control blood samples. Transcriptional data were validated in additional advanced-stage mycosis fungoides/Sézary syndrome skin and blood samples by immunofluorescence microscopy. RESULTS: Several nonoverlapping clonotypes are expanded in the skin and blood of individual advanced-stage mycosis fungoides/Sézary syndrome patient samples, including a dominant malignant clone as well as additional minor malignant and reactive clones. While we detected upregulation of patient-specific as well as mycosis fungoides- and Sézary syndrome-specific oncogenic pathways within individual malignant clones, we also detected upregulation of several common pathways that included genes associated with cancer cell metabolism, cell-cycle regulation, de novo nucleotide biosynthesis, and invasion. CONCLUSIONS: Our analysis unveils new insights into mycosis fungoides/Sézary syndrome pathogenesis by providing an unprecedented report of the transcriptional profile of malignant T-cell clones in the skin and blood of individual patients and offers novel prospective targets for personalized therapy.

Single-cell transcriptome analysis identifies skin-specific T-cell responses in systemic sclerosis.

OBJECTIVES: Although T cells have been implicated in the pathogenesis of systemic sclerosis (SSc), a comprehensive study of T-cell-mediated immune responses in the affected skin of patients with progressive SSc is lacking. Droplet-based single-cell transcriptome analysis of SSc skin biopsies opens avenues for dissecting patient-specific T-cell heterogeneity, providing a basis for identifying novel gene expression related to functional pathways associated with severity of SSc skin disease. METHODS: Single-cell RNA sequencing was performed by droplet-based sequencing (10x Genomics), focusing on 3729 CD3+ lymphocytes (867 cells from normal and 2862 cells from SSc skin samples) from skin biopsies of 27 patients with active SSc and 10 healthy donors. Confocal immunofluorescence microscopy of progressive SSc skin samples validated transcriptional results and visualised spatial localisations of T-cell subsets. RESULTS: We identified several subsets of recirculating and tissue-resident T cells in healthy and SSc skin that were associated with distinct signalling pathways. While most clusters shared a common gene expression signature between patients and controls, we identified a unique cluster of recirculating CXCL13+ T cells in SSc skin which expressed a T helper follicular-like gene expression signature and that appears to be poised to promote B-cell responses within the inflamed skin of patients. CONCLUSIONS: Current available therapies to reverse or even slow progression of SSc lead to broad killing of immune cells and consequent toxicities, including death. Identifying the precise immune mechanism(s) driving SSc pathogenesis could lead to innovative therapies that selectively target the aberrant immune response, resulting in better efficacy and less toxicity.

Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma.

The signal transducer and activator of transcription 6 (STAT6) is a critical up-stream mediator of interleukin-13 (IL-13) and IL-4 signaling and is constitutively activated in malignant lymphocytes from Sezary syndrome (SS) and mycosis fungoides (MF), the most common subtypes of cutaneous T-cell lymphomas. By combining genome-wide expression profiling with pharmacological STAT6 inhibition, we have identified the genes regulated by STAT6 in MF/SS tumors. We found that STAT6 regulates several common pathways in MF/SS malignant lymphocytes that are associated with control of cell-cycle progression and genomic stability as well as production of Th2 cytokines. Using ex vivo skin explants from cutaneous MF tumors as well as Sezary cells derived from the blood of SS patients, we demonstrated that inhibition of STAT6 activation downregulates cytokine production and induces cell-cycle arrest in MF/SS malignant lymphocytes, inhibiting their proliferation but not their survival. Furthermore, we show that STAT6 promotes the protumoral M2-like phenotype of tumor-associated macrophages in the tumor microenvironment of advanced stage MF by upregulating the expression of genes associated with immunosuppression, chemotaxis, and tumor matrix remodeling. Thus, we show STAT6 to be a major factor in the pathogenesis and progression of MF/SS, promoting proliferation and invasion of the malignant lymphocytes while inducing a progressive depression of the antitumor immune response. Together, our results provide new insights into disease pathogenesis and offer new prospective targets for therapeutic intervention.

Single-Cell Lymphocyte Heterogeneity in Advanced Cutaneous T-cell Lymphoma Skin Tumors.

PURPOSE: The heterogeneity of tumor cells presents a major challenge to cancer diagnosis and therapy. Cutaneous T-cell lymphomas (CTCL) are a group of T lymphocyte malignancies that primarily affect skin. Lack of highly specific markers for malignant lymphocytes prevents early diagnosis, while only limited treatment options are available for patients with advanced stage CTCL. Droplet-based single-cell transcriptome analysis of CTCL skin biopsies opens avenues for dissecting patient-specific T lymphocyte heterogeneity, providing a basis for identifying specific markers for diagnosis and cure of CTCL. EXPERIMENTAL DESIGN: Single-cell RNA-sequencing was performed by Droplet-based sequencing (10X Genomics), focusing on 14,056 CD3+ lymphocytes (448 cells from normal and 13,608 cells from CTCL skin samples) from skin biopsies of 5 patients with advanced-stage CTCL and 4 healthy donors. Protein expression of identified genes was validated in advanced stage CTCL skin tumors by immunohistochemistry and confocal immunofluorescence microscopy. RESULTS: Our analysis revealed a large inter- and intratumor gene expression heterogeneity in the T lymphocyte subset, as well as a common gene expression signature in highly proliferating lymphocytes that was validated in multiple advanced-stage skin tumors. In addition, we established the immunologic state of reactive lymphocytes and found heterogeneity in effector and exhaustion programs across patient samples. CONCLUSIONS: Single-cell analysis of CTCL skin tumor samples reveals patient-specific landscapes of malignant and reactive lymphocytes within the local microenvironment of each tumor, giving an unprecedented view of lymphocyte heterogeneity and identifying tumor-specific molecular signatures, with important implications for diagnosis and personalized disease treatment.

T cells and cytokines in systemic sclerosis.

PURPOSE OF REVIEW: Dysregulation of both the innate and the adaptive immune systems has been identified in systemic sclerosis (SSc). However, the mechanisms underlying aberrant immune cell function remain poorly understood. T cells represent a predominant cell type in the affected tissues of patients, particularly in the early inflammatory stage of the disease. Antigen specificity of infiltrating T cells has not been identified; however, recent studies implicate specific T-cell subsets and the cytokines they produce in SSc pathogenesis by modulating the development of autoimmunity, inflammation and fibrosis. RECENT FINDINGS: The phenotype and function of distinct T-cell subsets have been identified in the affected tissues of SSc patients as well as in SSc animal models, implying their contribution to disease process. The molecular mechanisms underlying cytokine dysregulation by specific T-cell subpopulations are also becoming clear. SUMMARY: A better understanding of SSc pathogenesis will allow the development of novel therapeutic strategies targeting specific cell types and the pathways that are abnormally activated as well as the cytokines produced that may be directly involved with disease process. A further goal is to tailor therapy to address dysregulation specific to individual patients, leading to better efficacy and reduced toxicity.

Current perspectives on the role of CD8+ T cells in systemic sclerosis.

Despite long-standing recognition of the importance of T cells in systemic sclerosis (SSc; scleroderma), the role of CD8+ T cells in disease pathogenesis has not been well studied. Our work has shown that over-production of the pro-fibrotic cytokine IL-13 by peripheral blood effector/memory CD8+ T cells is critical for predisposing patients to more severe forms of cutaneous fibrosis. Moreover, IL-13-producing CD8+ T cells induce a pro-fibrotic phenotype in normal and SSc dermal fibroblasts, and exhibit a strong cytotoxic activity ex vivo. We also found that CD8+ T cells are predominantly abundant in the skin lesions of patients in the early stages of diffuse cutaneous (dc)SSc compare to late-stage disease patients. Isolation of CD8+ T cells from the lesional skin of early active dcSSc patients, established that they are skin-resident, express cytolytic molecules and co-express extremely high levels of IL-13 and IFNγ. Other recent studies corroborate these findings and together strongly suggest that CD8+ T cells contribute to SSc pathogenesis through the production of high levels of cytokines with pro-inflammatory and pro-fibrotic function as well as by exhibiting a cytotoxic activity.

14-3-3z sequesters cytosolic T-bet, upregulating IL-13 levels in TC2 and CD8+ lymphocytes from patients with scleroderma.

BACKGROUND: IL-13-producing CD8+ T cells have been implicated in the pathogenesis of type 2-driven inflammatory human conditions. We have shown that CD8+IL-13+ cells play a critical role in cutaneous fibrosis, the most characteristic feature of systemic sclerosis (SSc; scleroderma). However, the molecular mechanisms underlying production of IL-13 and other type 2 cytokines by CD8+ T cells remain unclear. OBJECTIVE: We sought to establish the molecular basis of IL-13 overproduction by CD8+ T cells from patients with SSc, focusing on T-bet modulation of GATA-3 activity, which we showed to underlie IL-13 overproduction in CD8+IL-13+ cells from patients with SSc. METHODS: Biochemical and biophysical methods were used to determine the expression and association of T-bet, GATA-3, and regulatory factors in CD8+ T cells isolated from the blood and lesional skin of patients with SSc with severe skin thickening. Chromatin immunoprecipitation analysis determined GATA-3 binding to the IL-13 promoter. ImageStream analysis and confocal microscopy visualized the subcellular localization of T-bet and GATA-3. Transcript levels were decreased by small interfering RNAs. RESULTS: Interaction of T-bet with the adaptor protein 14-3-3z in the cytosol of CD8+ T cells from patients with SSc reduces T-bet translocation into the nucleus and its ability to associate with GATA-3, allowing more GATA-3 to bind to the IL-13 promoter and inducing IL-13 upregulation. Strikingly, we show that this mechanism is also found during type 2 polarization of CD8+ T cells (TC2) from healthy donors. CONCLUSIONS: We identified a novel molecular mechanism underlying type 2 cytokine production by CD8+ T cells, revealing a more complete picture of the complex pathway leading to SSc disease pathogenesis.

Skin-Resident Effector Memory CD8+CD28- T Cells Exhibit a Profibrotic Phenotype in Patients with Systemic Sclerosis.

Loss of CD28 expression by CD8+ T cells occurs with age and during chronic inflammatory conditions. CD8+CD28- T cells are a heterogeneous cell subpopulation whose function ranges from immunosuppressive to effector. Here we analyzed the role of CD8+CD28- T cells in the pathogenesis of systemic sclerosis (SSc), a connective tissue disorder characterized by autoimmunity, vasculopathy, and extensive cutaneous and visceral fibrosis. We show that the frequency of CD8+CD28- T cells is increased in the blood and affected skin of SSc patients, independent of patient age, and correlates with the extent of skin fibrosis. We found that most skin-tropic CD8+CD28- T cells are resident in the skin lesions of patients in the early stage of the disease, exhibit an effector memory phenotype, and present a strong cytolytic activity ex vivo. Skin-resident and circulating SSc CD8+CD28- T cells produce high levels of the profibrotic cytokine IL-13, which induces collagen production by normal and SSc dermal fibroblasts. Thus, our findings indicate that CD8+CD28- T cells represent a pathogenic T-cell subset in SSc and likely play a critical role in the early stage of SSc skin disease.

Current perspectives on the immunopathogenesis of systemic sclerosis.

Systemic sclerosis (SSc or scleroderma) is a progressive and highly debilitating autoimmune disorder characterized by inflammation, vasculopathy, and extensive fibrosis. SSc is highly heterogeneous in its clinical presentation, extent and severity of skin and internal organ involvement, and clinical course and has the highest fatality rate among connective tissue diseases. While clinical outcomes have improved in recent years, no current therapy is able to reverse or slow the natural progression of SSc, a reflection of its complex pathogenesis. Although activation of the immune system has long been recognized, the mechanisms responsible for the initiation of autoimmunity and the role of immune effector pathways in the pathogenesis of SSc remain incompletely understood. This review summarizes recent progress in disease pathogenesis with particular focus on the immunopathogenetic mechanisms of SSc.

Interleukin-13 is overexpressed in cutaneous T-cell lymphoma cells and regulates their proliferation.

Cutaneous T-cell lymphomas (CTCLs) primarily affect skin and are characterized by proliferation of mature CD4(+) T-helper cells. The pattern of cytokine production in the skin and blood is considered to be of major importance for the pathogenesis of CTCLs. Abnormal cytokine expression in CTCLs may be responsible for enhanced proliferation of the malignant cells and/or depression of the antitumor immune response. Here we show that interleukin-13 (IL-13) and its receptors IL-13Rα1 and IL-13Rα2 are highly expressed in the clinically involved skin of CTCL patients. We also show that malignant lymphoma cells, identified by the coexpression of CD4 and TOX (thymus high-mobility group box), in the skin and blood of CTCL patients produce IL-13 and express both receptors. IL-13 induces CTCL cell growth in vitro and signaling through the IL-13Rα1. Furthermore, antibody-mediated neutralization of IL-13 or soluble IL-13Rα2 molecules can lead to inhibition of tumor-cell proliferation, implicating IL-13 as an autocrine factor in CTCL. Importantly, we established that IL-13 synergizes with IL-4 in inhibiting CTCL cell growth and that blocking the IL-4/IL-13 signaling pathway completely reverses tumor-cell proliferation. We conclude that IL-13 and its signaling mediators are novel markers of CTCL malignancy and potential therapeutic targets for intervention.

Interleukin-13-producing CD8+ T cells mediate dermal fibrosis in patients with systemic sclerosis.

OBJECTIVE: Fibrosis is a major contributor to morbidity and mortality in systemic sclerosis (SSc). T cells are the predominant inflammatory infiltrate in affected tissue and are thought to produce cytokines that drive the synthesis of extracellular matrix (ECM) proteins by fibroblasts, resulting in excessive fibrosis. We have previously shown that aberrant interleukin-13 (IL-13) production by peripheral blood effector CD8+ T cells from SSc patients correlates with the extent of skin fibrosis. The present study was undertaken to investigate the role of IL-13 production by CD8+ T cells in dermal fibrosis, an early and specific manifestation of SSc. METHODS: ECM protein production by normal dermal fibroblasts cocultured with SSc CD8+ T cell supernatants was determined by quantitative polymerase chain reaction and Western blotting. Skin-homing receptor expression and IL-13 production by CD8+ T cells in the peripheral blood of SSc patients were measured by flow cytometry. IL-13+ and CD8+ cells in sclerotic skin were identified by immunohistochemistry. RESULTS: IL-13-producing circulating CD8+ T cells from patients with SSc expressed skin-homing receptors and induced a profibrotic phenotype in normal dermal fibroblasts, which was inhibited by an anti-IL-13 antibody. High numbers of CD8+ T cells and IL-13+ cells were found in the skin lesions of SSc patients, particularly during the early inflammatory phase of the disease. CONCLUSION: These findings show that IL-13-producing CD8+ T cells are directly involved in modulating dermal fibrosis in SSc. The demonstration that CD8+ T cells homing to the skin early in the course of SSc are associated with accumulation of IL-13 is an important mechanistic contribution to the understanding of the pathogenesis of dermal fibrosis in SSc and may represent a potential target for therapeutic intervention.

Role of IL-13 in systemic sclerosis.

Systemic sclerosis (SSc) has the highest fatality rate among connective tissue diseases and is characterized by vascular damage, inflammation and fibrosis. Currently, no therapy has proven effective in modifying the course of SSc, a reflection of its complex pathogenesis. T cell-derived cytokines have been implicated in the induction of fibrosis. The role of the pro-fibrotic type 2 cytokine IL-13 and its regulation appear to be important in the pathogenesis of SSc and other fibrotic disorders. Recent work has shown that dysregulated production of IL-13 by effector CD8+ T cells is critical for predisposing patients to more severe forms of cutaneous disease and that this dysregulation is associated with defects in the molecular control of IL-13 production, such as increased expression of the transcription factor GATA-3. Silencing of GATA-3 with siRNA significantly reduces IL-13 production by CD8+ T cells from patients. We review these new insights into SSc pathogenesis that will enable establishment of highly relevant biomarkers of immune dysfunction in patients predisposed to develop SSc and open new possibilities for development of more specific diagnosis and treatment.

CD8+ T cells in systemic sclerosis.

Systemic sclerosis (SSc) is a progressive and highly debilitating autoimmune disorder characterized by inflammation, fibrosis, and vascular damage of the connective tissue. T cell-derived cytokines have been implicated in the induction of fibrosis. We found that high levels of the profibrotic type-2 cytokine IL-13 are produced by peripheral blood effector CD8(+) T cells from SSc patients compared to normal controls. This abnormality correlates with increased expression of the transcription factor GATA-3 and the extent of skin fibrosis. Together, the data provide new insights into SSc pathogenesis and identify a specific T cell phenotype that can be used as a biomarker of immune dysfunction in patients with SSc and as a novel therapeutic target for this currently incurable disease.

GATA-3 up-regulation in CD8+ T cells as a biomarker of immune dysfunction in systemic sclerosis, resulting in excessive interleukin-13 production.

OBJECTIVE: Despite the importance of interleukin-13 (IL-13) in systemic sclerosis (SSc) and other fibrotic diseases, its mechanisms of action are not understood. We have reported that excessive amounts of IL-13 are produced by peripheral blood effector CD8+ T cells from patients with diffuse cutaneous SSc (dcSSc). The aim of the present study was to establish the molecular basis of IL-13 dysregulation in the pathogenesis of SSc. METHODS: Quantitative polymerase chain reaction analysis and intracellular staining were used to study the transcription factors that control naive peripheral blood CD8+ T cell differentiation into type 1 and type 2 cytokine-secreting cells. Intracellular staining revealed that GATA-3 levels in freshly isolated naive CD8+ T cells correlated with specific clinical manifestations. We therefore assessed the effects of GATA-3 inhibition on IL-13 production in CD8+ T cells from the SSc patients. RESULTS: Freshly isolated naive peripheral blood CD8+ T cells expressed high levels of GATA-3 and failed to down-regulate IL-13 production when cultured under type 1-skewing conditions, but maintained adequate levels of interferon-γ production. Cellular GATA-3 levels were significantly higher in patients with dcSSc and early inflammatory disease. Silencing of GATA-3 with small interfering RNA significantly reduced IL-13 production by CD8+ T cells, demonstrating a causal relationship between GATA-3 and IL-13. CONCLUSION: These results provide important new insights into SSc pathogenesis and suggest that increased GATA-3 expression in CD8+ T cells could be a highly relevant biomarker of immune dysfunction in patients with dcSSc. GATA-3 could be a novel therapeutic target for this currently incurable disease.

Gene expression analysis of dendritic cells that prevent diabetes in NOD mice: analysis of chemokines and costimulatory molecules.

We have demonstrated previously that BM-derived DCs can prevent diabetes development and halt progression of insulitis in NOD mice, the mouse model of type 1 diabetes. The DC population that was most effective in this therapy had a mature phenotype, expressed high levels of costimulatory molecules, and secreted low levels of IL-12p70. The protective DC therapy induced Treg and Th2 cells in vitro and in vivo. Microarray analysis of therapeutic and nontherapeutic DC populations revealed differences in the expression of OX40L, CD200, Ym-1, CCL2, and CCL5, which could play important roles in the observed DC-mediated therapy. The unique pattern of costimulatory molecules and chemokines expressed by the therapeutic DCs was confirmed by flow cytometry and ELISA. Using a novel cell-labeling and (19)F NMR, we observed that the chemokines secreted by the therapeutic DCs altered the migration of diabetogenic Th1 cells in vivo and attracted Th2 cells. These results suggest that the therapeutic function of DCs is mediated by a combination of costimulatory and chemokine properties that results in the attraction of diabetogenic Th1 and the induction of Th2 and/or Treg differentiation.