Worawidh Wajjwalku

A novel autoregulatory mechanism for transcriptional activation of the IL-15 gene by a nonsecretable isoform of IL-15 generated by alternative splicing

There are several isoforms of interleu kin (IL) ‐15 generated by alternating splicing. We reported previously that alternative IL‐15 transgenic (Tg) mice expressing an IL‐15 cDNA isoform encoding nonsecretable IL‐15 protein had an impaired ability to produce IL‐15. In this study, we found that expression of endogenous IL‐15 mRNA but not tumor necrosis factor a mRNA was severely impaired in response to lipopolysaccharide, not only in macrophages from al ternative IL‐15 Tg mice but also in RAW264.7 cells that had been transfected with alternative IL‐15 together with IL‐15 receptor α (IL‐15Rα). IL‐15 promoter activ ity was suppressed in the transfected cells. Although nuclear factor‐kB activation was not impaired, the binding activity of nuclear extracts to the interferon‐ stimulated response element of the IL‐15 promoter region was reduced in RAW264.7 cells, which had been cotransfected with alternative IL‐15 and IL‐15Rα. IL‐15 was mainly colocalized with IL‐15Rα at the cytoplasmic membrane of RAW264.7 cells, which had been cotrans fected with normal IL‐15, whereas nonsecretable IL‐15 was colocalized with IL‐15Rα in nucleus after cotrans fection with alternative IL‐15 and IL‐15Rα. These re sults suggest that nonsecretable IL‐15 generated by alternative splicing suppresses further IL‐15 gene tran scription, implying a novel autocrine regulatory mech anism for cytokine gene expression by alternative splicing.

Overexpression of interleukin‐15 in vivo enhances antitumor activity against mhc class I‐negative and ‐positive malignant melanoma through augmented NK activity and cytotoxic T‐cell response

Interleukin (IL)‐15, a pleiotropic cytokine, is involved in the development and maintenance of NK cells and memory CD8+ T cells. We examined the effects of in vivo overexpression of IL‐15 on protection against 2 types of murine B16 melanoma lines, MHC class I‐negative B16.44 and MHC class I‐positive B16F10 cells, using IL‐15 transgenic (Tg) mice that we have recently constructed. The tumor growth was severely retarded in IL‐15 Tg mice after subcutaneous (s.c.) inoculation with B16.44 or B16F10 cells. IL‐15 Tg mice showed an augmented NK cell activity against B16.44 cells, and in vivo depletion of NK cells by anti‐asialoGM1 Ab treatment abrogated the antitumor activity in IL‐15 Tg mice. On the other hand, IL‐15 Tg mice inoculated with B16F10 cells developed a significant level of CTL response against B16F10 cells, and in vivo depletion of CD8+ T cells by anti‐CD8 MAb treatment abrogated the antitumor activity. Thus, overexpression of IL‐15 augmented antitumor activity against different tumors via augmentation of different antitumor mechanisms. These results suggest a possible therapeutic application of IL‐15 for human neoplasms expressing a wide range of MHC class molecules.

Overexpression of Interleukin-15 Protects against Escherichia coli-Induced Shock Accompanied by Inhibition of Tumor Necrosis Factor-α-Induced Apoptosis

Interleukin (IL)-15, a potent inhibitor of tumor necrosis factor (TNF)-alpha-mediated apoptosis, causes multiple organ failure during endotoxic shock. We investigated the potential role of IL-15 in protection against Escherichia coli-induced shock by using IL-15 transgenic (Tg) mice. These mice were resistant to an otherwise lethal challenge with E. coli, although bacterial burden and serum levels of TNF-alpha were similar in non-Tg mice. Apoptosis in cells of the peritoneal cavity, liver, spleen, or lung was significantly suppressed in IL-15 Tg mice after E. coli infection. Peritoneal cells from naive IL-15 Tg mice were also resistant to TNF-alpha-induced apoptosis in vitro, and neutralization of endogenous IL-15 significantly aggravated TNF-alpha-induced apoptosis. Exogenous IL-15 prevented TNF-alpha-induced apoptosis in normal mice in vitro and improved the survival rate after E. coli challenge. These results suggest that IL-15 overexpression can prevent TNF-alpha-induced apoptosis and protect against E. coli-induced shock, indicating a possible therapeutic application of IL-15 for septic shock.

Efficacy of Recombinant Bacille Calmette-Guérin Vaccine Secreting Interleukin-15/Antigen 85B Fusion Protein in Providing Protection against Mycobacterium tuberculosis

Protection against Mycobacterium tuberculosis not only depends on CD4+ T helper type 1 (Th1) cells but, also, on CD8+ T cells. Interleukin (IL)-15 has an important function in the maintenance of memory CD8+ T cells. In the present study, we examined the efficacy of recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) secreting fusion protein antigen (Ag) 85B murine IL-15 (rBCG-Ag85B-IL15) in providing protection against M. tuberculosis infection. The levels of major histocompatibility (MHC) class Ib (H2-M3)-binding TB2- or MHC class Ia (H-2Db)-binding MPT64-specific CD8+ T cells producing interferon (IFN)-gamma were significantly higher after immunization with rBCG-Ag85B-IL15 than after immunization with rBCG secreting Ag85B (rBCG-Ag85B). The levels of purified protein derivative- or Ag85B-specific CD4+ T cells producing IFN-gamma were also higher in mice immunized with rBCG-Ag85B-IL15 than in mice immunized with rBCG-Ag85B. Mice immunized with rBCG-Ag85B-IL15 exhibited CD8+ and CD4+ T cells responses that were stronger than those in mice immunized with rBCG-Ag85B, as well as robust protection in the lung against intratracheal challenge of M. tuberculosis. Thus, rBCG-Ag85B-IL15 vaccination capable of inducing efficient cell-mediated immunity might be used as an effective vaccine for tuberculosis.

A Novel Role of CD30L/CD30 Signaling by T-T Cell Interaction in Th1 Response against Mycobacterial Infection

A CD30 ligand (CD30L, CD153) is a type II membrane-associated glycoprotein belonging to the TNF family. To illustrate the potential role of CD30L in CD4+ Th1 cell responses, we investigated the fate of Ag-specific CD4+ T cells in CD30L-deficient (CD30L−/−) mice after Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. The number of bacteria was significantly higher in organs of CD30L−/− mice than in wild-type (WT) mice 4 wk postinfection. The numbers of purified protein derivative- or Ag85B-specific-IFN-γ-producing-CD4+ T cells in spleen, lung, or peritoneal exudate cells were significantly fewer in CD30L−/− mice than in WT mice. During the infection, CD30L was expressed mainly by CD44+CD3+CD4+ T cells but not by CD3+CD8+ T cells, B cells, dendritic cells, or macrophages. Costimulation with agonistic anti-CD30 mAb or coculturing with CD30L-transfected P815 cells restored IFN-γ production by CD4+ T cells from BCG-infected CD30L−/− mice. Coculturing with CD30L+/+CD4+ T cells from BCG-infected WT mice also restored the number of IFN-γ+CD30L−/−CD4+ T cells. When transferred into the CD30L+/+ mice, Ag-specific donor CD30L−/− CD4+ T cells capable of producing IFN-γ were restored to the compared level seen in CD30L+/+ CD4+ T cells on day 10 after BCG infection. When naive CD30L+/+ T cells were transferred into CD30L−/− mice, IFN-γ-producing-CD4+ Th1 cells of donor origin were normally generated following BCG infection, and IFN-γ-producing-CD30L−/−CD4+ Th1 cells of host origin were partly restored. These results suggest that CD30L/CD30 signaling executed by CD30+ T-CD30L+ T cell interaction partly play a critical role in augmentation of Th1 response capable of producing IFN-γ against BCG infection.

Y-chromosomal variation confirms independent domestications of swamp and river buffalo

Y-chromosomal variation in the water buffalo was analysed by sequencing of DBY, ZFY and SRY gene segments. A clear separation of the paternal lineages of the river and swamp types parallels the differences between their maternal lineages and nuclear DNA. Sequence divergence was found to be comparable to the divergence of taurine cattle and zebu, and this divergence predated domestication, confirming that river and swamp buffalo originated from different wild populations. Within a sample of 23 Thai swamp buffaloes, we identified four haplotypes with different geographical distributions, two of which were shared by Thai wild buffaloes.

IL-15 protects antigen-specific CD8+ T cell contraction after Mycobacterium bovis bacillus Calmette-Guérin infection

We reported previously that IL‐15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following acute infection with Listeria monocytogenes by inducing antiapoptotic molecules. In the present study, we examined the effects of in vivo administration of rIL‐15 on contraction of CD8+ T cells after chronic infection with Mycobacterium bovis BCG and on the efficacy of BCG vaccination against Mycobacterium tuberculosis infection. Antigen‐specific CD8+ T cells reached an expansion peak at approximately Day 21, followed by a contraction after inoculation with rBCG expressing OVA. In vivo administration of rIL‐15 from Days 22 to 42 after BCG inoculation inhibited apoptosis of effector CD8+ T cells by up‐regulating their Bcl‐2 expression, resulting in a significant increase of antigen‐specific memory CD8+ T cells producing IFN‐γ. However, the IL‐15 treatment did not elicit improved efficacy of BCG vaccination against M. tuberculosis. These results suggest that IL‐15 plays a critical role in protecting activated CD8+ T cells from apoptosis during the contraction phase following BCG inoculation, although IL‐15 administration alone at the contraction phase might not be sufficient to protect the efficient memory T cell responses against subsequent infection with M. tuberculosis.

H2-M3-Restricted CD8+ T Cells Induced by Peptide-Pulsed Dendritic Cells Confer Protection against Mycobacterium tuberculosis

One of the oligopolymorphic MHC class Ib molecules, H2-M3, presents N-formylated peptides derived from bacteria. In this study, we tested the ability of an H2-M3-binding peptide, TB2, to induce protection in C57BL/6 mice against Mycobacterium tuberculosis. Immunization with bone marrow-derived dendritic cell (BMDC) pulsed with TB2 or a MHC class Ia-binding peptide, MPT64190–198 elicited an expansion of Ag-specific CD8+ T cells in the spleen and the lung. The number of TB2-specific CD8+ T cells reached a peak on day 6, contracted with kinetics similar to MPT64190–198-specific CD8+ T cells and was maintained at an appreciable level for at least 60 days. The TB2-specific CD8+ T cells produced less effector cytokines but have stronger cytotoxic activity than MPT64190–198-specific CD8+ T cells. Mice immunized with TB2-pulsed BMDC as well as those with MPT64190–198-pulsed BMDC showed significant protection against an intratracheal challenge with M. tuberculosis H37Rv. However, histopathology of the lung in mice immunized with TB2-pulsed BMDC was different from mice immunized with MPT64190–198-pulsed BMDC. Our results suggest that immunization with BMDC pulsed with MHC class Ib-restricted peptides would be a useful vaccination strategy against M. tuberculosis.

First whole genome characterization of swine influenza virus subtype H3N2 in Thailand

H3N2 swine influenza viruses (SIV) were first detected in Asia shortly after the 1968 pandemic emerged in humans. Subsequently, human H3N2 viruses have sporadically reappeared in swine. In Thailand, a human-like H3N2 SIV was reported in 1978 although the genetic sequence of this virus is unknown. In this study, we undertook cross sectional syndromic surveillance in pigs in four provinces in Thailand. Seven genetically similar H3N2 viruses were isolated. A representative, A/SW/Thailand/KU5.1/04, was fully sequenced and shown to contain genes from human-like influenza viruses and North American and European SIV. The results restate that transmission of influenza A virus among human and swine populations is common and that genes from both American and Eurasian SIV lineages cocirculate in Thailand.

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y‐chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y‐chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.