Distinctive Pattern of Cytokine Production and Adhesion Molecule Expression in Peripheral Blood Memory CD4+ T Cells from Patients with Active Crohn’s Disease

An expansion of both circulating and intestinal lamina propria CD4⁺CD45RO⁺ T cells has been described in patients with Crohn’s disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4⁺CD45RO⁺ T cells from patients with Crohn’s disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4⁺CD45RO⁺ T cells producing intracellular TNF was higher in active than in inactive Crohn’s disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn’s disease patients was also found. CD62L was downregulated in active Crohn’s disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4⁺CD45RO⁺ T cells from active Crohn’s disease lesions showed an increased intracellular staining of TNF, IFN-γ, and IL-10. Both peripheral and intestinal mucosa CD4⁺CD45RO⁺ T cells from active Crohn’s disease patients show an increased production of TNF. In addition, the circulating CD4⁺CD45RO⁺ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn’s disease.Dr. García de Tena and Dr. Manzano are joint first authors     

CD44 Deficiency Contributes to Enhanced Experimental Autoimmune Encephalomyelitis: A Role in Immune Cells and Vascular Cells of the Blood–Brain Barrier

Adhesion molecule CD44 is expressed by multiple cell types and is implicated in various cellular and immunological processes. In this study, we examined the effect of global CD44 deficiency on myelin oligodendrocyte glycoprotein peptide (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Compared to C57BL/6 wild-type mice, CD44-deficient mice presented with greater disease severity, increased immune cell numbers in the central nervous system, and increased anti-MOG antibody and proinflammatory cytokine production, especially those associated with T helper 17 (Th17) cells. Further, decreased numbers of peripheral CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) were observed in CD44-knockout mice throughout the disease course. CD44-knockout CD4 T cells exhibited reduced transforming growth factor-β receptor type I (TGF-β RI) expression that did not impart a defect in Treg polarization in vitro, but did correlate with enhanced Th17 polarization in vitro. Further, EAE in bone marrow–chimeric animals suggested CD44 expression on both circulating and noncirculating cells limited disease severity. Endothelial expression of CD44 limited T-cell adhesion to and transmigration through murine endothelial monolayers in vitro. Importantly, we also identified increased permeability of the blood–brain barrier in vivo in CD44-deficient mice before and following immunization. These data suggest that CD44 has multiple protective roles in EAE, with effects on cytokine production, T-cell differentiation, T-cell–endothelial cell interactions, and blood–brain barrier integrity.Multiple sclerosis (MS) is an autoimmune, demyelinating disease resulting from chronic inflammation in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE), the primary and long-used animal model of MS, produces immune processes relevant to the human disease.¹ The progression and pathogenesis of EAE is complex and depends on multiple cell types and processes.^2–4T helper 17 (Th17) cells and their distinctive cytokine, IL-17, play pivotal roles in EAE/MS pathogenesis.^5–7 Th17 cells, members of a CD4 T-cell effector subset, are generated from naive CD4 T-cell precursors in response to cytokines TGF-β and IL-6, whereas IL-23 expands this population and increases pathogenicity.^8,9 In EAE, Th17 cells first infiltrate and initiate recruitment to the CNS,^5,6 and Th17-produced IL-17 induces neuronal death⁶ and increases permeability of the blood–brain barrier (BBB), allowing continued influx of immune cells by disrupting endothelial cell (EC) junctions.^6,10Regulatory T cells (Tregs), the primary suppressors of the immune system, play a pivotal role in EAE that is opposite to Th17 cells. Treg depletion exacerbates disease symptoms, whereas supplementation with additional Tregs ameliorates the disease.^11,12 Identified by the expression pattern CD4⁺CD25⁺FoxP3⁺, Tregs are generally divided into two principal subsets: naturally occurring Tregs, which arise in the thymus during development, and induced Tregs (iTregs), which can be generated in the periphery from naive CD4 T cells in response to TGF-β.^13,14Vascular EC also contribute to the complex pathogenesis of EAE. EC regulate leukocyte adhesion and extravasation, maintain vascular integrity, and limit injury and immune-mediated vascular permeability. The CNS vasculature, the primary constituent of the BBB, is especially unique and plays a critical role in protecting the CNS microenvironment. In MS/EAE, there is a characteristic breakdown of the BBB followed by accumulation of inflammatory infiltrates.^15,16CD44, a ubiquitously expressed type I transmembrane glycoprotein, has been implicated in a wide variety of cellular processes within and outside of the immune system.^17,18Alternative splicing and multiple posttranslational modifications generate various structural and functional versions of CD44 and are thought to be responsible for its large range of diverse and sometimes seemingly contradictory cellular functions.Although CD44 has been studied in several immunological contexts as a positive or negative regulator of inflammation, the many results are confounded by use of different mouse strains, inflammatory models, and experimental approaches. CD44 has been implicated as a proinflammatory molecule in several studies that identified an anti-inflammatory effect of a CD44 monoclonal antibody in multiple immune-mediated processes and diseases such as lymphocyte extravasation,¹⁹collagen- or proteoglycan-induced arthritis, respectively,^20,21 type 1 diabetes,²² asthma,²³ and EAE.²⁴ However, most studies in CD44-knockout (KO) mice suggest an anti-inflammatory role for this molecule in various immunological processes instead. CD44-KO mice experience enhanced inflammation in several models of pulmonary inflammation that suggest various roles of CD44 in immune cell clearance, TGF-β signaling, and repression of Toll-like receptor (TLR) signaling and inflammatory gene expression.^25–28 Further, CD44-KO mice show increased septic responses to lipopolysaccharide²⁹ and enhanced inflammatory responses following myocardial infarction³⁰ or hepatic injury.³¹ CD44 deficiency also led to increased collagen-induced arthritis severity with up-regulation of inflammatory genes in arthritic CD44-KO T cells.³² Clearly, antibody-mediated interference can have very different effects than genetic disruption of CD44. Hutas et al³³ in 2008 reported disparate effects of CD44 monoclonal antibody treatment versus CD44 deficiency on leukocyte recruitment during proteoglycan-induced arthritis.Despite antibody-mediated interference studies, the role of CD44 in EAE/MS remains poorly understood. In active MS lesions, there is an increase in CD44 expression and accumulation of hyaluronan (HA), a major CD44 ligand.^34,35 Previously, CD44 was shown to facilitate uptake of HA,³⁶ promoting resolution of tissue-injury signals and inflammation.^25,37 By contrast, a conditional mouse model of oligodendrocyte-specific overexpression of CD44 found a correlation between CD44 expression and enhanced HA accumulation, prevention of oligodendrocyte differentiation, and subsequent inflammation-independent demyelination.³⁵Until recently, EAE had not been examined in CD44-KO mice. This report demonstrates that CD44-KO mice present with increased EAE disease severity. This was associated with loss of CD44 on circulating immune cells, and also on noncirculating cells, specifically vascular EC of the BBB. We illustrate a more proinflammatory T-cell profile in CD44-KO mice with a reduction in Treg numbers throughout the disease that is accompanied by increased permeability of the BBB. Further, we illustrate a previously unidentified role for CD44 in the baseline integrity of the BBB, which has far-reaching implications beyond MS/EAE.     

CD4+CD25+CD127low/? T Cells: A More Specific Treg Population in Human Peripheral Blood

The quantitative identification and enrichment of viable regulatory T cells (Treg) requires reliable surface markers that are selectively expressed on Treg. Foxp3 is the accepted marker of nTreg, but it cannot be used to isolate cells for functional studies. In this study, we compared four staining profiles of Treg, including CD4⁺CD25^high T cells, CD4⁺CD39⁺ T cells, CD4⁺CD73⁺ T cells, and CD4⁺CD25⁺CD127^low/? T cells. We found that CD4⁺CD25⁺CD127^low/? T cells expressed the highest level of Foxp3 and had the strongest correlation with CD4⁺CD25⁺Foxp3⁺ T cells, the accepted identifying characteristics for ??real?? nTreg cells. Moreover, functional data showed that CD4⁺CD25⁺CD127^low/? T cells could effectively suppress the proliferation of CD4⁺CD25^? T cells, suggesting that compared with the other three populations, CD4⁺CD25⁺CD127^low/? T cells best fit the definition of naturally occurring regulatory T cells in human peripheral blood. Finally, we showed that CD4⁺CD25⁺CD127^low/? can be used to quantitate Treg cells in individuals with systemic lupus erythematosus supporting the use of CD4⁺CD25⁺CD127^low/? to identify human Treg cells.     

CD4+CD7− T Cells: A Separate Subpopulation of Memory T Cells?

The CD7 molecule is apparently involved in T cell activation but is absent in a substantial subpopulation of human T cells under physiological and certain pathological conditions. The majority of CD7^– T cells expresses TCR / and is of CD4⁺ helper and CD45R0⁺CD45RA^– memory phenotype. After birth, percentages and absolute numbers of circulating CD7^– T cells increase significantly during aging. A number of molecules thought to be involved in organ-specific T cell homing are preferentially expressed within the subset of CD4⁺CD7^– T cells. Specific absence of CD7 antigen expression on T cells is observed in a variety of pathologic conditions such as cutaneous T cell lymphoma, HIV infection, rheumatoid arthritis, and kidney transplantation. Current in vitro results suggest that specific downregulation of CD7 antigen expression in T cells reflects a separate and stable differentiation state occurring late in the immune response. Expansion of CD7^– T cells in vivo has been found in certain diseases associated with chronically repeated T cell stimulation. The potential pathophysiological significance of this T cell subset in certain human diseases is discussed.     

Quantitative Peripheral Blood Perturbations of γδ T Cells in Human Disease and Their Clinical Implications

Human γδ T cells, which play innate and adaptive, protective as well as destructive, roles in the immune response, were discovered in 1986, but the clinical significance of alterations of the levels of these cells in the peripheral blood in human diseases has not been comprehensively reviewed. Here, we review patterns of easily measurable changes of this subset of T cells in peripheral blood from relevant publications in PubMed and their correlations with specific disease categories, specific diagnoses within disease categories, and prognostic outcomes. These collective data suggest that enumeration of γδ T cells and their subsets in the peripheral blood of patients could be a useful tool to evaluate diagnosis and prognosis in the clinical setting.     

Role of Different CD40 Polymorphisms in Graves’ Disease and Hashimoto’s Thyroiditis

Genome-wide association studies have led to the discovery of several susceptibility genes related to autoimmune thyroid diseases (AITDs). However, controversial results have been reported regarding the role of single-nucleotide polymorphism (SNP) of CD40 in the disease susceptibility. The objective of this study was to identify the relationship of the polymorphisms of three sites of CD40 with the susceptibility to AITD in the Chinese population. We genotyped three polymorphisms of CD40: C/T ?1 SNP, 58038T site of the third exon and C64610G site of the ninth exon in 196 GD cases, 121 HT cases and 122 control subjects. The three putative polymorphism sites were amplified by PCR for sequencing and analysis. The genotype frequencies of CD40 ?1 C/C genotype and C allele were significantly higher in the GD group than those in normal control. For the C64610G polymorphism, the C/G genotype was significantly more frequent in HT group than in control group, and the G allele frequencies in the GD and HT group were both higher than those in control group. These results indicated that there exist different susceptibility loci for AITD within CD40, each contributing a different effect in the onset and development of AITDs.     

CD4+LAP+ and CD4+CD25+Foxp3+ Regulatory T Cells Induced by Nasal Oxidized Low-Density Lipoprotein Suppress Effector T Cells Response and Attenuate Atherosclerosis in ApoE−/− Mice

Increasing studies have demonstrated that atherosclerosis is a chronic immunoinflammatory disease, and that oxidized low-density lipoprotein (oxLDL)-specific T cells contribute to the autoimmune process in atherosclerosis. Oral administration of oxLDL, which was identified as a candidate autoantigen in atherosclerosis, was shown to induce tolerance and suppress atherogenesis. However, the precise mechanisms of mucosal tolerance induction, in particular nasal tolerance, remain unknown. In this study, we explored the effect of nasal oxLDL on atherosclerosis as well as the cellular and molecular mechanisms leading to atheroprotective responses, and then found that nasal oxLDL drastically ameliorate the initiation (47.6 %, p?p?=?0.001) of atherosclerosis. Most importantly, a significant 35.8 % reduction of the progression of atherosclerosis was observed in the enhanced immunization group (p?+ latency-associated peptide (LAP)⁺ regulatory T cells (Tregs) and CD4⁺CD25⁺Foxp3⁺ Tregs in spleens and cervical lymph nodes, together with increased transforming growth factor (TGF)-β production and suppressed T-helper cells type 1, 2, and 17 immune responses. Surprisingly, neutralization of TGF-β in vivo partially counteracted the protective effect of nasal oxLDL treatment, indicating that the presence of TGF-β was indispensable to CD4⁺LAP⁺ Tregs and CD4⁺CD25⁺Foxp3⁺ Tregs to acquire regulatory properties. Our studies suggest that CD4⁺LAP⁺ Tregs and CD4⁺CD25⁺Foxp3⁺ Tregs induced by nasal delivery of oxLDL can inhibit oxLDL-specific T cells response and ameliorate atherosclerosis process.     

CD4+ T Cells and IFN-γ Are Required for the Development of Pneumocystis-Associated Pulmonary Hypertension

Pulmonary hypertension (PH) is a disease of diverse etiology. Although primary PH can develop in the absence of prior disease, PH more commonly develops in conjunction with other pulmonary pathologies. We previously reported a mouse model in which PH occurs as a sequela of Pneumocystis infection in the context of transient CD4 depletion. Here, we report that instead of the expected Th2 pathways, the Th1 cytokine IFN-γ is essential for the development of PH, as wild-type mice developed PH but IFN-γ knockout mice did not. Because gene expression analysis showed few strain differences that were not immune-function related, we focused on those responses as potential pathologic mechanisms. In addition to dependence on IFN-γ, we found that when CD4 cells were continuously depleted, but infection was limited by antibiotic treatment, PH did not occur, confirming that CD4 T cells are required for PH development. Also, although CD8 T-cells are implicated in the pathology of Pneumocystis pneumonia, they did not have a role in the onset of PH. Finally, we found differences in immune cell phenotypes that correlated with PH, including elevated CD204 expression in lung CD11c⁺ cells, but their role remains unclear. Overall, we demonstrate that a transient, localized, immune response requiring IFN-γ and CD4-T cells can disrupt pulmonary vascular function and promote lingering PH.Pulmonary hypertension (PH) is a devastating disease with complex etiology and, in all likelihood, diverse mechanisms of pathology. A recent reclassification of the types of PH involves five major divisions, including forms associated with specific causative agents (such as drugs), hypoxia, and infectious agents (such as schistosomes).¹ A common feature of many of these agents is that they initiate local inflammation, which may act as a trigger for the development of PH,^2–4 even if the inflammation does not persist after the manifestation of PH. However, there does not seem to be any single inflammatory mediator responsible for the inflammatory initiation of PH. For example, several immune cell types (T cells, B cells, and macrophages) and inflammatory cytokines (TGF-β, IL-1β, IL-6, RANTES, and IL-13) have been implicated in various forms of PH.^5–9In the T helper 1 and T helper 2 (Th1 and Th2) paradigm, CD4⁺ Th2 cells drive an immune response characterized by the production of cytokines such as IL-4, IL-5, and IL-13, as well as by secreted antibody (in particular, IgE).¹⁰ In several studies using animal models, a strong case has been made for a role of Th2 immune responses as instigators of PH. For example, a Th2 response associated with sensitization to an antigen and subsequent challenge with that antigen can result in muscularization of smaller pulmonary arteries, and this response is associated with CD4⁺ cells and IL-13.¹¹ The protein resistin-like alpha (Retnla; alias cysteine-rich secreted protein FIZZ1) can be induced by hypoxia (which is associated with vascular remodeling¹²), but it is also induced in Th2 immune responses; in some cases, the Th2-associated molecule Retn1a appears to have a strong association with vascular remodeling and resultant PH,^13,14 which may be related to its induction by hypoxia, itself a potent stimulator of vascular remodeling.¹⁴ An interesting mouse model of PH associated with repeated inhalation of spores of the fungus Stachybotrys chartarum also is associated with the Th2 cytokines IL-4 and IL-5, but not the Th1 cytokine IFN-γ.¹⁵ Finally, in what is probably one of the best-known examples of PH in conjunction with an infectious agent, schistosomiasis-induced PH appears to be associated with the Th2 cytokine IL-13.¹⁶ Furthermore, IL-13 is implicated in several other forms of PH.¹⁷In contrast, Th1 immune responses, which are characterized by the secretion of cytokines such as IFN-γ and TNF-α and activation of phagocytic macrophages, appear to have little connection with the development of vascular remodeling and PH. Despite a few reports of elevated TNF-α in conjunction with clinical syndromes that include PH,^7,18 there is very little association of PH with the canonical Th1 cytokine, IFN-γ, although there is one report of IFN-γ having a synergistic effect with other cytokines on in vitro pulmonary vascular cell remodeling.¹⁹ Indeed, this lack of effect is illustrated by the fact that IFN-γ has been used in clinical treatment of idiopathic pulmonary fibrosis,²⁰ although with little effectiveness, even though ≤40% of patients with idiopathic pulmonary fibrosis also exhibit PH.²¹Recently, we reported that PH developed in the aftermath of a resolved Pneumocystis pneumonia in mice, in the context of a transient depletion of CD4⁺ cells.²² At the time, it was unclear which immune responses are involved in the development of PH in that mouse model; although there were elevated levels of Retnla (FIZZ1) in the bronchoalveolar lavage fluid (BALF) of mice that developed PH, as well as some perivascular fibrosis, IL-4 signaling was not required for these developments.²² In the present study, surprisingly, we found that the Th1 cytokine IFN-γ is absolutely required for the development of PH in this mouse model, and that perivascular fibrosis does not appear to be a cause of PH. Furthermore, having previously established that onset of PH in these mice is correlated with the resurgence of CD4⁺ T cells after depletion,²² with the present study we have demonstrated that CD4⁺ cells are also absolutely required for the development of PH.     

Association of Graves’ Disease and Prevalence of Circulating IFN-γ-producing CD28<Superscript>−</Superscript> T Cells

Background  Peripheral blood CD4⁺ and CD8⁺ T-cell subsets lacking surface CD28 have been suggested to predispose patients to immune-mediated disorders. Materials and Methods  To determine the role of CD28⁻ T-cell subset in Graves’ disease (GD), we characterized peripheral blood CD4⁺CD28⁻ and CD8⁺CD28⁻ T cell from early onset GD patients. Results and Discussion  GD patients had significantly higher percentages of CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells than did healthy donors. Both CD28⁻ T cells expressed mostly CD45RO, suggesting that they are activated and/or are memory T cells. GD patient-derived CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells produced more intracellular IFN-γ than their counterparts from healthy donors. Furthermore, CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells from GD patients with Graves’ ophthalmopathy (GO) secreted higher level of intracellular IFN-γ than those CD28⁻ T cells from GD patients without GO. Retrospective analysis showed that the increased levels of CD4⁺CD28⁻ T cells and their IFN-γ-producing subgroups were positively correlated to the serum anti-thyrotropin receptor (TSHR) autoantibodies (TRAb). Our observations suggest that increased IFN-γ-producing CD28⁻ T cells in GD patients may play an important role in the pathogenesis of GD. Zhiping Sun and Weixue Zhong contributed equally to this paper.     

Bacillus Calmette-Guérin Suppresses Asthmatic Responses via CD4+CD25+ Regulatory T Cells and Dendritic Cells

Purpose

Bacillus Calmette-Guérin (BCG) is known to suppress the asthmatic responses in a murine model of asthma and to induce dendritic cells (DCs) maturation. Mature DCs play a crucial role in the differentiation of regulatory T cells (Tregs), which are known to regulate allergic inflammatory responses. To investigate whether BCG regulates Tregs in a DCs-mediated manner, we analyzed in a murine model of asthma.

Methods

BALB/c mice were injected intraperitoneally with BCG or intravenously with BCG-stimulated DCs and then sensitized and challenged with ovalbumin (OVA). Mice were analysed for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. To identify the mechanisms, IgE, IgG₁ and IgG_2a in the serum were analysed and the CD25₊ Tregs in the mice were depleted with anti-CD25 monoclonal antibody (mAb).

Results

BCG and the transfer of BCG-stimulated DCs both suppressed all aspects of the asthmatic responses, namely, BHR, the production of total IgE and OVA-specific IgE and IgGs, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment reversed these effects.

Conclusions

BCG can attenuate the allergic inflammation in a mouse model of asthma by a Tregs-related mechanism that is mediated by DCs.     

2-Gy whole-body irradiation significantly alters the balance of CD4+CD25? T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice

CD4⁺CD25⁺ T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4⁺CD25⁺Foxp3⁺ Treg cells has not been determined. The proportion, phenotypes and function of CD4⁺CD25⁺ Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁺Foxp3⁺ Treg cells to CD4⁺ T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4⁺CD25⁺ Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4⁺CD25⁻ T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4⁺CD25⁺Foxp3⁺ Treg cells than in CD4⁺CD25⁻ T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.     

Trypanosoma cruzi-Induced Activation of Functionally Distinct αβ and γδ CD4? CD8? T Cells in Individuals with Polar Forms of Chagas' Disease

CD4⁻ CD8⁻ (double-negative [DN]) T cells have recently been shown to display important immunological functions in human diseases. They express γδ or αβ T-cell receptors that recognize lipid/glycolipid antigens presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently demonstrated that while αβ DN T cells serve primarily to express inflammatory cytokines, γδ DN T cells express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi experimental infection. In this work, we sought to investigate whether αβ or γδ DN T cells display distinct immunoregulatory potentials in patients with polar forms of human Chagas'' disease. Our data showed that in vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe cardiac clinical forms of the disease. However, while αβ DN T cells primarily produce inflammatory cytokines in both forms of the disease, γδ DN T cells display a marked, significant increase in antigen-specific IL-10 expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-producing γδ DN T cells were correlated with improved clinical measures of cardiac function in the patients, suggesting a protective role for these cells in Chagas'' disease. Taken together, these data show distinct functional characteristics for αβ and γδ DN T cells associated with distinct morbidity rates and clinical forms in human Chagas'' disease.T-cell activation is a key event in the establishment of immune responses directed toward intracellular pathogens. Depending on the functional capacity of the activated T cells, the fate of the infection may take different paths either toward a protective or a pathogenic outcome. While it is important that a strong, activated immune response is elicited early on in the infection in order to eliminate (or control) the pathogen, the further control of this activation is necessary to reestablish homeostasis, avoiding tissue damage (17, 25).One hallmark of most parasitic infections is that the great majority of individuals are able to trigger innate immunity and elicit an activated T-cell response during the acute infection, leading to the control of the parasite and establishment of a chronic infection. Interestingly, while many individuals develop severe forms of parasitic diseases once infection progresses to the chronic phase, most patients develop relatively mild forms, allowing for a host-parasite coexistence. One such example is observed upon human infection with the protozoan parasite Trypanosoma cruzi, which leads to Chagas'' disease. As a result of thousands of years of coevolution between human host and the parasite (6), most infected individuals develop an asymptomatic, or “indeterminate” (I), form of Chagas'' disease. This form is characterized by a lack of clinical signs and symptoms and has been associated predominantly with a modulatory cellular immune response based on cytokine profiles and downregulatory molecule expression (5, 20, 48, 49, 51). Chronic patients may also develop symptomatic clinical forms, mainly with digestive or cardiac alterations. Differential geographical prevalence of Chagas'' disease clinical forms has been reported. In Brazil, 15 to 30% of Chagas'' patients display the cardiac form, which is present in 20 states, while the digestive cases, observed in about 10% of infected individuals, have been reported in four states in the central region of the country (53). The digestive form is frequently found in Chile but is practically absent in Central America (42). These geographical differences might be related, in part, to host genetics and immune responses of local human populations, but it is believed that they are also related to the genetic diversity of T. cruzi strains (11). Different strains of parasite display tropism for different tissues, and, thus, an important factor determining the clinical course of disease might be the specific pool of infecting clones and their specific tropisms (29). However, a possible role for environmental, nutritional, and immunological aspects of the host cannot be discounted. While digestive and cardiac forms present significant morbidity, the cardiac form is the one associated with highest mortality. It is caused by neuronal and cardiomyocyte damage, ultimately resulting in ventricular dilation and subsequent functional heart failure, which can lead to death (44). Cardiac patients display a T-cell-mediated inflammatory response in situ (13, 24, 41), which is responsible for the pathology; this inflammatory profile is also observed in circulating activated T cells found at high frequencies in these patients (2, 16, 19, 32). Although it is clear that a plethora of parasite and host factors influences the clinical outcome of Chagas'' disease, recent studies have suggested that activation of functionally distinct T-cell populations in T. cruzi-infected individuals may be responsible for the establishment of different clinical forms (17, 20). Thus, identifying these populations and the factors responsible for their activation will be critical for driving immune-based interventions to prevent pathology.While the great majority of T cells express either the CD4 or the CD8 molecules, which are important for stabilizing the peptide-major histocompatibility complex (MHC) complex and which favor T-cell activation, a minority population of T cells that do not express CD4 or CD8 molecules has been identified in humans (8, 10, 27, 37). These double-negative (DN) T cells have been shown to be important sources of immunoregulatory cytokines in human leishmaniasis (4), to display modulatory functions (38), but also, under different circumstances, to display cytolytic activity (10, 36). A subpopulation of DN T cells is activated through the engagement of αβ or γδ T-cell receptors (TCRs) in the recognition of nonclassical MHC molecules of the CD1 family, presenting lipid or glycolipid antigens (36). This particular lipid/glycolipid antigenic recognition, as well as the immunoregulatory potential and susceptibility to chronic stimulation of these cells, highlights the important role these cells play in parasitic infections.In our work with Bottrel et al., we determined that DN lymphocytes were the second most prevalent cell type producing gamma interferon (IFN-γ) in human cutaneous leishmaniasis and that this IFN-γ production was seen after short-term cultures with medium alone, as well as after stimulation with soluble Leishmania antigen (SLA) (9). The novel work of Antonelli et al. went on to demonstrate that DN T cells composed of two different cell populations are present in the blood of individuals infected with Leishmania braziliensis and that DN T cells expressing the αβ TCR displayed a profile consistent with activation of leishmanicidal and inflammatory activities (higher IFN-γ and tumor necrosis factor alpha [TNF-α]) while the DN subpopulation expressing γδ TCR had a modulatory potential via higher production of interleukin-10 (IL-10) (4). Interestingly, IFN-γ production has been associated with pathogenic responses in human leishmaniasis in more than one clinical form (3, 7, 22). We recently demonstrated that rats infected with the CL-Brenner clone of T. cruzi displayed a marked increase in the frequency of circulating DN T cells during the acute phase of infection (33). Taken together, these data led to the question of the role that DN T-cell subpopulations play in the clinical dichotomy of chronic human Chagas'' disease.To answer these questions, we investigated the immunoregulatory potential of DN T cells in patients with the two polar forms of Chagas'' disease: indeterminate (I) and dilated cardiac (DC). Our data demonstrated that although no quantitative differences were seen with regard to the nonstimulated frequency of DN αβ and γδ T-cell subpopulations between patients and nonchagasic individuals, in vitro infection with trypomastigote forms of T. cruzi induced a marked increase in the frequency of these cells from chagasic patients. Moreover, the expanded αβ DN T cells displayed a greater inflammatory potential from cardiac patients than from indeterminate patients. This was accompanied by a greater down-modulatory ratio of IL-10 to inflammatory cytokine frequencies by γδ DN T cells from individuals with indeterminate disease, suggesting distinct roles for these cells in modulating the response in chronic Chagas'' disease. Finally, we observed a correlation between higher frequencies of IL-10-producing γδ DN T cells and improved clinical measures of cardiac function, suggesting a protective role for these cells in human Chagas'' disease. These data indicate that functionally distinct DN T cells are present in Chagas'' disease patients and that they are associated with the resulting morbidity of the disease.