Material Supplementary 4.DC1http://www.jimmunol.org/content/suppl/2010/02/22/jimmunol.090332

The earliest thymic progenitors (ETPs) were recently shown to give rise to both lymphoid and myeloid cells. While the majority of ETPs are derived from IL-7Rα-positive cells and give rise exclusively to T cells, the origin of the myeloid cells remains undefined. Herein, we show both in vitro and in vivo that IL-13Rα1+ ETPs yield myeloid cells with no potential for maturation into T cells while IL-13Rα1− ETPs lack myeloid potential. Moreover, transfer of lineage-negative IL-13Rα1+ bone marrow stem cells into IL-13Rα1-deficient mice reconstituted thymic IL-13Rα1+ myeloid ETPs. Myeloid cells or macrophages in the thymus are regarded as phagocytic cells whose function is to clear apoptotic debris generated during T cell development. However, the myeloid cells derived from IL-13Rα1+ ETPs were found to perform antigen presenting functions. Thus, IL-13Rα1 defines a new class of myeloid restricted ETPs yielding Ag presenting cells that could contribute to development of T cells and the control of immunity and autoimmunity.

Summary In this study we examined the role IL-13 receptor alpha 1 (IL-13Rα1) plays in macrophage differentiation and function. The findings indicate that IL-13Rα1 is expressed on the M2 but not the M1 subset of macrophages and specifically heterodimerizes with the IL-4Rα chain to form a type II receptor, which controls the differentiation and function of these cells. Indeed, bone marrow (BM) cells from IL-13Rα1+/+ and IL-13Rα1−/− mice yield equivalent numbers of macrophages when cultured under M2 polarizing conditions. However, IL-13Rα1−/− BM cells yield a much higher number of macrophages than IL-13Rα1+/+ BM cells when the differentiation is carried out under M1-polarizing conditions. Further analyses indicated that macrophages that express IL-13Rα1 also display surface markers associated with an M2 phenotype. In addition, the IL-13Rα1+ macrophages were highly efficient in phagocytizing zymosan bioparticles both in vitro and in vivo, and supported differentiation of naïve T cells to a Th2 phenotype. Finally, when stimulated by IL-13, a cytokine that uses the heteroreceptor, the cells were able to phosphorylate STAT6 efficiently. These previously unrecognized findings indicate that IL-13Rα1 serves as a marker for M2 macrophages and the resulting heteroreceptor influences both their differentiation and function.

Neonatal immunity exhibits weak Th1 but excessive Th2 responses and the underlying mechanisms remain elusive. Here, we show that neonatal basophils readily produce IL-4, a cytokine that proved to be pivotal in shaping the programs of both lymphocyte subsets. Besides promoting Th2 programs, IL-4 is captured by the IL-4 heteroreceptor (IL-4Rα/IL-13Rα1) expressed on dendritic cells and instigates IL-12 down-regulation. Under these circumstances, differentiating Th1 cells up-regulate IL-13Rα1 leading to an unusual expression of the heteroreceptor which will serve as a death marker for these Th1 cells during re-challenge with antigen (Ag). The resulting Th1/Th2 imbalance impacts childhood immunity culminating in sensitivity to allergic reactions, susceptibility to microbial infection and perhaps poor efficacy of pediatric vaccines.

Type 1 diabetes involves both T helper (Th)1 and Th17 cells. While the mechanisms underlying the control of Th1 cells are relatively well defined, those operating modulation of Th17 cells remain unknown. Moreover, given that Th17 cells are plastic and can drive disease as stable or convertible T cells, effective approaches to counter type 1 diabetes would have to alter Th17 function under both circumstances. Herein, we genetically incorporated the BDC2.5-reactive p79 mimotope into an Ig molecule, and the resulting Ig-p79 was used to investigate Th17 tolerance. Accordingly, diabetogenic BDC2.5 Th17 cells were transferred into NOD mice under convertible or stable conditions and their fate was evaluated upon induction of tolerance and disease suppression by Ig-p79. The findings show that convertible (Th17 to Th1) cells display downregulation of the chemokine (C-X-C motif) receptor 3 that was associated with diminished T-box transcription factor T-bet expression, retention in the spleen, and inhibition of trafficking to the pancreas. In contrast, stable Th17 cells downregulated orphan nuclear receptor ROR-γt but increased Fas ligand expression and died by apoptosis. Thus, the final signature transcription factor shapes the mechanism of tolerance in plastic Th17 cells. These findings suggest that effective strategies against type 1 diabetes will require regimens that could drive both mechanisms of tolerance to overcome the disease.

Upon exposure to Ag on the day of birth, neonatal mice mount balanced primary Th1 and Th2 responses with the former displaying up-regulated IL-13 receptor alpha 1 (IL-13Rα1) expression. This chain associates with IL-4Rα to form a heteroreceptor (IL-4Rα/IL-13Rα1) that marks the Th1 cells for death by IL-4 produced by Th2 cells during re-challenge with Ag, hence, the Th2 bias of murine neonatal immunity. The up-regulation of IL-13Rα1 on neonatal Th1 cells was due to the paucity of IL-12 in the neonatal environment. Herein, we show that by day 8 after birth, naïve splenic T cells are no longer susceptible to IL-13Rα1 up-regulation even when exposed to Ag within the neonatal environment. Furthermore, during the 8-day lapse, the naïve splenic T cells spontaneously and progressively up-regulate the IL-12Rβ2 chain, perhaps due to colonization by commensals which induce production of IL-12 by cells of the innate immune system such as dendritic cells. In fact, mature T cells from the thymus, a sterile environment not accessible to microbes, did not up-regulate IL-12Rβ2 and were unable to counter IL-13Rα1 up-regulation. Finally, the 8 day naïve T cells were able to differentiate into Th1 cells even independently of IL-12 but required the cytokine to counter up-regulation of IL-13Rα1. Thus, in neonatal mice, IL-12, which accumulates in the environment progressively, utilizes IL-12Rβ2 to counter IL-13Rα1 expression in addition to promoting Th1 differentiation.

The role APCs play in the transition of T cells from effector to memory remains largely undefined. This is likely due to the low frequency at which long-lived T cells arise, which hinders analysis of the events involved in memory development. In this study, we used TCR transgenic T cells to increase the frequency of long-lived T cells and developed a transfer model suitable for defining the contribution of APCs to the development of CD4 T cell memory. Accordingly, naive TCR transgenic T cells were stimulated in vitro with Ag presented by different types of APCs and transferred into MHC class II-deficient mice for parking, and the hosts were later analyzed for long-lived T cell frequency or challenged with suboptimal dose of Ag, and the long-lived cells-driven memory responses were measured. The findings indicate that B cells and CD8α+ dendritic cells sustained elevated frequencies of long-lived T cells that yielded rapid and robust memory responses upon rechallenge with suboptimal dose of Ag. Furthermore, both types of APCs had significant programmed death (PD) ligand 2 expression prior to Ag stimulation, which was maintained at a high level during presentation of Ag to T cells. Blockade of PD ligand 2 interaction with its receptor PD-1 nullified the development of memory responses. These previously unrecognized findings suggest that targeting specific APCs for Ag presentation during vaccination could prove effective against microbial infections.

ABSTRACT. In Nepal, mycobacterial isolates obtained from the milk and feces of buffaloes and cattle that were positive for the single intradermal cervical tuberculin (SICT) tests were genetically identified. A total of 36 mycobacterial strains were isolated from 39% of the buffaloes (14 of 36) and 34% of the cattle (11 of 32). Of the 36 strains, 13 were identified as M. bovis, and these strains were isolated from 17% of the buffaloes (6 of 36) and 16% of the cattle (5 of 32). M. bovis was isolated from both the milk and feces of one buffalo and one cattle, the milk alone of three buffaloes and three cattle, and the feces alone of two buffaloes and one cattle. These results suggest that milking buffaloes and cattle infected with M. bovis exist in Nepal. The remaining 23 strains were atypical mycobacteria. A program for the elimination of bovine tuberculosis should be implemented as soon as possible, and the public health education and proper hygienic practices may be required. KEY WORDS: buffalo, cattle, isolation, mycobacteria, Nepal.J. Vet. Med. Sci. 69(8): 819-825, 2007 In humans and animals, mycobacteria such as those belonging to the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. canettii, and M. microti) cause serious diseases, and atypical mycobacteria cause opportunistic infections. Many species of mycobacteria have been identified and it has been suggested that many unknown atypical mycobacteria species exist [10,20,24]. In developing countries, M. tuberculosis is one of the most common causes of human tuberculosis. In humans, however, M. bovis infection exists and a large population of unknown causes may be attributable to M. bovis [2]. In addition, ruminant animals such as cattle and buffaloes are thought to be the reservoirs of M. bovis [2,3,5,9,18,19]. Previously, we had examined the results of the single intradermal cervical tuberculin (SICT) test was carried out by injection tuberculin into the skin of the neck of a large number of cattle and buffaloes in Nepal. We found that approximately 5% of these animals had a history of M. bovis infection, and that food products from these animals were being consumed daily in the country [21]. However, this study did not determine the M. bovis strain that was responsible for the infection.Recently, mycobacterial species were genetically identified by using rpoB gene analysis [10,11]. In addition, M. tuberculosis and M. bovis were identified by using the hypothetical protein "Rv1506c" [1]. In this study, we applied a genetic method such as sequence homology of the rpoB gene to the identification of the species of the isolates, and investigated the prevalence of mycobacteria in the milk and feces of SICT test-positive milking buffaloes and cattle. We also performed sequence and phylogenetic analyses of their rpoB gene. MATERIALS AND METHODS Samples:From September to October in 2003, we collected the milk and fecal samples from 36 buffaloes and 32 cattle that were SICT test-positive. The animals were selected from the Kathmand...