Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4(+) T lymphocytes

The costimulatory receptors CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 and their ligands, CD80 and CD86, are expressed on T lymphocytes; however, their functional roles during T cell-T cell interactions are not well known. The consequences of blocking CTLA-4-CD80/CD86 interactions on purified mouse CD4(+) T cells were studied in the context of the strength of signal (SOS). CD4(+) T cells were activated with phorbol 12-myristate 13-acetate (PMA) and different concentrations of a Ca2+ ionophore, Ionomycin (I), or a sarcoplasmic Ca2+ ATPase inhibitor, Thapsigargin (TG). Increasing concentrations of I or TG increased the amount of interleukin (IL)-2, reflecting the conversion of a low to a high SOS. During activation with PMA and low amounts of I, intracellular concentrations of calcium ([Ca2+](i)) were greatly reduced upon CTLA-4-CD80/CD86 blockade. Further experiments demonstrated that CTLA-4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of I or TG (high SOS) but the opposite occurred with PMA and low amounts of I or TG (low SOS). These results were confirmed by surface T-cell receptor (TCR)-CD3 signalling using a low SOS, for example soluble anti-CD3, or a high SOS, for example plate-bound anti-CD3. Also, CTLA-4-CD80/CD86 interactions enhanced the generation of reactive oxygen species (ROS). Studies with catalase revealed that H2O2 was required for IL-2 production and cell cycle progression during activation with a low SOS. However, the high amounts of ROS produced during activation with a high SOS reduced cell cycle progression. Taken together, these results indicate that [Ca2+](i) and ROS play important roles in the modulation of T-cell responses by CTLA-4-CD80/CD86 interactions.

Effect of Temperature Variation on Sister Chromatid Exchange Frequency in Cultured Human Lymphocytes

The effect of temperature variation on sister chromatid exchange (SCE) frequencies in human lymphocytes was studied. An increase as well as decrease in incubation temperature of cells leads to a higher frequency of sister chromatid exchanges than in cultures grown at 37°C. In addition, it was observed that mitotic: index and cell cycle duration were affected by low temperature.

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group, JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis, H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV, Virus replication was found to be inhibited in the brains of animals that mere adoptively transferred with JEV specific CTL as revealed by immunohistological staining as,veil as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.

Glycodelin-A interferes with IL-2/IL-2R signalling to induce cell growth arrest, loss of effector functions and apoptosis in T-lymphocytes

The progesterone-regulated glycoprotein glycodelin-A (GdA), secreted by the decidualized endometrium at high concentrations in primates, inhibits the maternal immune response against fetal antigens and thereby contributes to the tolerance of the semi-allogenic fetus during a normal pregnancy. Our earlier studies demonstrated the ability of GdA to induce an intrinsic apoptotic cascade in CD4 T-lymphocytes and suppress the cytolytic effector function of CD8 T-lymphocytes. In this report, we investigated further into the mechanism of action of GdA controlling perforin and granzyme B expression in CD8 T-lymphocytes and the mechanism of action of GdA leading to lymphocyte death. Flow cytometry analysis was performed to check for the surface expression of interleukin-2 receptor (IL-2R) and intracellular eomesodermin (Eomes) in activated T-lymphocytes, whereas quantitative RTPCR analysis was used to find out their mRNA profile upon GdA treatment. Western analysis was carried out to confirm the protein level of Bax and Bcl-2. GdA reduces the surface expression of the high-affinity IL-2R complex by down-regulating the synthesis of IL-2R (CD25). This disturbs the optimal IL-2 signalling and decreases the Eomes expression, which along with IL-2 directly regulates perforin and granzymes expression. Consequently, the CD8 T-lymphocytes undergo growth arrest and are unable to mature into competent cytotoxic T-lymphocytes. In the CD4 T-lymphocytes, growth factor IL-2 deprivation leads to proliferation inhibition, decreased Bcl-2/enhanced Bax expression, culminating in mitochondrial stress and cell death. GdA spurs cell cycle arrest, loss of effector functions and apoptosis in different T-cell subsets by making T-lymphocytes unable to respond to IL-2.

Glycodelin A suppresses the cytolytic activity of CD8(+) T lymphocytes

Maternal tolerance to the semi-allogenic fetus is brought about by several mechanisms in humans Glycodelin A (GdA) secreted by the uterine mucosa and decidua is induced to high levels by progesterone between 12 and 16 weeks of pregnancy The glycoprotein an immunomodulator has been shown to be inhibitory to the survival and functions of almost all the immune cells CD8(+) T cells which predominate the T lymphocyte population in the decidua are relatively less studied We attempted to find out the possible mechanism if any of regulation of the cytolytic function of CD8(+) T cells during pregnancy Alloactivated CD8(+) T cells harbouring specific cytolytic activity against target cells exhibited compromised activity upon treatment with high concentrations of GdA Interestingly unlike the CD4(+) T cells CD8(+) T cells were resistant to GdA-induced apoptosis The inhibition of cytotoxic T lymphocyte activity was brought about by the downregulation of transcription of the cytolytic effector molecules granzyme B and perform and the degranulation of cytolytic vesicles These results suggest a protective role played by GdA during pregnancy by regulating the cytolytic activity of CD8(+) T cells (C) 2010 Elsevier Ltd All rights reserved

Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance.

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

Antitumor immunity in rats against Yoshida ascites sarcoma after treatment with antibiotics

Chromomycin A3, mithramycin, olivomycin and actinomycin D, four antitumor antibiotics inhibit growth of Yoshida ascites sarcoma (YAS). The antibiotic treated tumor-free rats exhibit antitumor immunity as judged by rejection of subsequent tumor transplant. The lymphocytes from immune rats are able to inhibit tumor growth in syngenic animals.

Recognition of nonstructural protein-peptides by cytotoxic t-lymphocytes raised against japanese encephalitis-virus

We have previously reported that Lyt(2+) cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K(d)-restricted CTL and to examine their cross-recognition of West Wile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K(d) binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.

Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: Requirement for L3T4(+) T cells

The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effecters by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effecters into newborn (4-day-old) and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2(+), L3T4(+) or Thy-1(+) T cell populations before adoptive transfer abrogated the protective ability of transferred effecters. However, when Lyt 2.2(+) cell-depleted and L3T4(+) cell-depleted effecters were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2(+) and L3T4(+) T cells are necessary to confer protection. Although the presence of L3T4(+) T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2(+) cells was not sufficient to confer protection.

Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs

Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a challenge with the virus of serotype A as well as O. This is the first report on the development of a bivalent FMD vaccine using a forage crop.

Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus

The Asian elephant Elephas maximus and the African elephant Loxodonta africana that diverged 5-7 million years ago exhibit differences in their physiology, behaviour and morphology. A comparative genomics approach would be useful and necessary for evolutionary and functional genetic studies of elephants. We performed sequencing of E. maximus and map to L. africana at similar to 15X coverage. Through comparative sequence analyses, we have identified Asian elephant specific homozygous, non-synonymous single nucleotide variants (SNVs) that map to 1514 protein coding genes, many of which are involved in olfaction. We also present the first report of a high-coverage transcriptome sequence in E. maximus from peripheral blood lymphocytes. We have identified 103 novel protein coding transcripts and 66-long non-coding (lnc)RNAs. We also report the presence of 181 protein domains unique to elephants when compared to other Afrotheria species. Each of these findings can be further investigated to gain a better understanding of functional differences unique to elephant species, as well as those unique to elephantids in comparison with other mammals. This work therefore provides a valuable resource to explore the immense research potential of comparative analyses of transcriptome and genome sequences in the Asian elephant.