Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD+CD25+ T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4+ population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4+CD25+ regulatory cells isolated in mice. With TCR cross-linking, CD4+CD25high cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4+CD25− responder T cells in a contact-dependent manner. The CD4+CD25high regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RBlow on murine CD4+CD25+ regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4+CD25high regulatory cells and CD4+CD25− responder cells. Whereas higher ratios of CD4+CD25high T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.

CD4+CD25+ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4+ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4+CD25+ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4+CD25hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4+CD25hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4+CD25hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4+CD25hi regulatory T cell function in patients with MS.

The recent discovery of CD4 + T cells characterized by secretion of interleukin (IL)-17 (T H 17 cells) and the naturally occurring regulatory FOXP3 + CD4 T cell (nT reg ) has had a major impact on our understanding of immune processes not readily explained by the T H 1/T H 2 paradigm. T H 17 and nT reg cells have been implicated in the pathogenesis of human autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis 1,2 . Our recent data and the work of others demonstrated that transforming growth factor-β (TGF-β) and IL-6 are responsible for the differentiation of naive mouse T cells into T H 17 cells, and it has been proposed that IL-23 may have a critical role in stabilization of the T H 17 phenotype [3][4][5] . A second pathway has been discovered in which a combination of TGF-β and IL-21 is capable of inducing differentiation of mouse T H 17 cells in the absence of ). However, TGF-β and IL-6 are not capable of differentiating human T H 17 cells 2,9 and it has been suggested that TGF-β may in fact suppress the generation of human T H 17 cells 10 . Instead, it has been recently shown that the cytokines IL-1β, IL-6 and IL-23 are capable of driving IL-17 secretion in short-term CD4 + T cell lines isolated from human peripheral blood 11 , although the factors required for differentiation of naive human CD4 to T H 17 cells are still unknown. Here we confirm that whereas IL-1β and IL-6 induce IL-17A secretion from human central memory CD4 + T cells, TGF-β and IL-21 uniquely promote the differentiation of human naive CD4 + T cells into T H 17 cells accompanied by expression of the transcription factor RORC2. These data will allow the investigation of this new population of T H 17 cells in human inflammatory disease.To better understand regulation of IL-17A secretion from human CD4 + T cells, we used a strategy that would allow us to evaluate the effects of various combinations of cytokine on expansion of T H 17 cells from memory T cells versus differentiation of naive CD4 + lymphocytes into T H 17 cells. Specifically, we used high-speed flow cytometry for sorting these two distinct populations of CD4 + T cells from the peripheral blood of healthy subjects: CD4 + CD25 − CD62L + CD45RA hi cells highly enriched for naive T cells and CD4 + CD25 − CD62L + CD45RA − cells enriched for central memory T cells (T CM ; Fig. 1a). All cells enriched for a naive or a central memory phenotype expressed the chemokine receptor CCR7 (data not shown). These two T cell populations were then stimulated with plate-bound

Multiple sclerosis (MS) is an autoimmune disease triggered by environmental factors that act on a genetically susceptible host. It features three clinical stages: a pre-clinical stage detectable only by MRI; a relapsing-remitting (RRMS) stage characterized by episodes of neurologic dysfunction followed by resolution; and a progressive stage, which usually evolves from the relapsing stage. Advances in our understanding of the immune mechanisms that contribute to MS have led to more than ten FDA-approved immunotherapeutic drugs that target effector T cells, regulatory cells, B cells, and cell trafficking into the nervous system. However, most drugs for relapsing MS are not effective in treating progressive disease. Progressive MS features a compartmentalized immune response in the central nervous system, involving microglia cells and astrocytes, as well as immune-independent processes that drive axonal dysfunction. Major challenges for MS research involve understanding the mechanisms of disease progression, developing treatment for progressive MS, and determining the degree to which progressive disease can be prevented by early treatment. Key priorities for MS research include developing biomarkers, personalized medicine and advanced imaging, and a better understanding of the microbiome. With a better understanding of the genetic and epidemiological aspects of this disease, approaches to prevent MS are now also being considered.

CD4+CD25highCD127low/− forkhead box p3 (Foxp3)+ regulatory T cells (Treg cells) possess functional plasticity. Here we describe a higher frequency of T helper type 1 (TH1)-like, interferon-γ (IFN-γ)-secreting Foxp3+ T cells in untreated subjects with relapsing remitting multiple sclerosis (RRMS) as compared to healthy control individuals. In subjects treated with IFN-β, the frequency of IFN-γ+Foxp3+ T cells is similar to that in healthy control subjects. In vitro, human Treg cells from healthy subjects acquire a TH1-like phenotype when cultured in the presence of interleukin-12 (IL-12). TH1-like Treg cells show reduced suppressive activity in vitro, which can partially be reversed by IFN-γ–specific antibodies or by removal of IL-12.

IntroductionRegulatory T cells (Tregs), characterized by high expression of FoxP3, are potent immunomodulators of T-cell activation that suppress proliferation and cytokine secretion by effector T cells. [1][2][3][4][5] Loss of Tregs by either gene deletion of FoxP3 in mice or mutations in humans results in autoimmune disease and the inability to effectively regulate T-cell activation. 6,7 While Tregs play a fundamental role in protection of autoimmunity, their differentiation is tightly linked to the development of IL-17-producing (Th17) cells, a highly pathogenic effector T-cell subset involved in inducing inflammation and autoimmune tissue injury. 8,9 Both loss of Treg function and induction of Th17 cells have been implicated in the pathogenesis of human autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, Crohn disease, and psoriasis. [10][11][12][13][14] Although Tregs appear to be central in regulating immune responses to self-antigens, mechanisms must be present to rapidly block their suppressive activity and enable immune activation during an acute microbial infection. In such circumstances, antigen-presenting cells (APC) secrete inflammatory cytokines, such as interleukin-1␤ (IL-1␤) and IL-6, which induce the potent effector responses necessary to clear the infection. In this regard, our data and the work of others have demonstrated that IL-1␤ and IL-6 can drive memory CD4 ϩ T cells to secrete 16 , which has been shown to abrogate suppression by Treg cells, 17 also drives Th17 differentiation in naive cells when paired with transforming growth factor ␤ (TGF␤) in mouse 18 or with IL-1␤, interleukin-23 (IL-23), and TGF␤ in human, 19 although the minimal requirements for Th17 differentiation in human are still being defined. 15,[20][21][22] The role of TGF␤ in Th17 development is surprising, given that TGF␤ promotes adaptive Treg differentiation in murine models and enhances FoxP3 expression in the human system. 23 Yet recent studies in mouse have indicated that TGF␤ and IL-6 can induce IL-17 production in Tregs. 24,25 Despite the apparent duality in Treg/Th17 function, it is becoming increasingly clear that these cell subsets share common cytokine signaling pathways.The memory CD4 ϩ CD25 high human Treg lineage can be subdivided into 2 functionally distinct subsets classified by expression of the major histocompatibility complex (MHC) class II dimer human leukocyte antigen-DR (HLA-DR). 26 DR ϩ Tregs do not enter into cell cycle with T-cell receptor (TCR) cross-linking and exhibit immediate contact-dependent suppressor function. In contrast, DR Ϫ Tregs can enter into cell cycle after activation, secrete the inhibitory cytokine interleukin-10 (IL-10) but not the effector cytokine interferon-␥ (IFN␥), and demonstrate delayed kinetics of suppression, requiring 4 to 5 days for maximal inhibition of proliferation of responder CD4 ϩ T cells. 26 We have previously reported that, in cocultures of human Tregs and responder T cells (Tresp) provided with strong TCR stimulation, the Tresp are refrac...

It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4+CD25high regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+CD25high T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II− CD4+CD25high Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25high Treg express higher levels of Foxp3 message and protein, compared with the DR−CD25high Treg population. Direct single-cell cloning of CD4+CD25high Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25high, and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25high identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.

Flow cytometric analysis allows rapid single cell interrogation of surface and intracellular determinants by measuring fluorescence intensity of fluorophore-conjugated reagents. The availability of new platforms, allowing detection of increasing numbers of cell surface markers, has challenged the traditional technique of identifying cell populations by manual gating and resulted in a growing need for the development of automated, high-dimensional analytical methods. We present a direct multivariate finite mixture modeling approach, using skew and heavy-tailed distributions, to address the complexities of flow cytometric analysis and to deal with high-dimensional cytometric data without the need for projection or transformation. We demonstrate its ability to detect rare populations, to model robustly in the presence of outliers and skew, and to perform the critical task of matching cell populations across samples that enables downstream analysis. This advance will facilitate the application of flow cytometry to new, complex biological and clinical problems.finite mixture model ͉ flow cytometry ͉ multivariate skew distribution F low cytometry transformed clinical immunology and hematology over 2 decades ago by allowing the rapid interrogation of cell surface determinants and, more recently, by enabling the analysis of intracellular events using fluorophore-conjugated antibodies or markers. Although flow cytometry initially allowed the investigation of only a single fluorophore, recent advances allow close to 20 parallel channels for monitoring different determinants (1-4). These advances have now surpassed our ability to interpret manually the resulting high-dimensional data and have led to growing interest and recent activity in the development of new computational tools and approaches (5-8).The difficulty in data analysis arises from the traditional technique of identifying discrete cell populations by manual gating, which is a labor-intensive process and varies by user experience. The initial computational packages for flow cytometric analyses focused largely on different preprocessing tasks such as data acquisition, normalization, and live cell gating. Besides visualization and transformation of flow cytometric data, useful tools such as Flowjo (www.flowjo.com) and the packages in BioConductor (www.bioconductor.org) (such as prada, flowCore, flowViz, flowUtils, and rflowcyt) allow some form of software-assisted gating and extraction of populations of interest. The operator subjectively demarcates a cell population while moving through successive 2-or 3-dimensional projections of the data. This process limits the reproducibility of data processing. A more fundamental problem is that this lower dimensional visualization hinders the identification of higher-dimensional features. Furthermore, current methods extract only a limited number of sample parameters, such as the mean fluorescence intensity of a cell population, which can lead to loss of critical information in defining the properties of a cell population....