Line L23 Research Articles

Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host’s immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.

Combining Ability Analysis of Maize Inbred Lines in Ethiopia

The study was initiated to estimate combining ability of maize inbred lines and crosses using line by tester analysis. Fifty entries consists 48 F1 single crosses developed from 24 inbred lines and 2 testers using line x tester design and two commercial check hybrids used in the study. The experiment was conducted using alpha lattice design with two replications. Analysis of variance revealed existence of significant genetic variation among genotypes for all studied traits except for plant aspect (PA). Location x entry interaction for most of the traits was not significant which suggests hybrid performance was consistent across tested locations. Line x tester analysis of variance showed that mean squares due to GCA of lines were significant (p< 0.01 or p< 0.05) for all studied traits. Mean squares of tester GCA and SCA were significant for most of studied traits. This indicates that both additive and non-additive gene effects had contributed for the variation of the crosses. However, higher proportional contribution of additive gene action for all studied traits was obtained. Several lines and crosses were identified as good general and specific combiners for yield and yield related traits. Lines L23, L11, L15 and crosses L2xT1, L3xT1, L8xT1, L11xT1, L23xT1 and L13xT2 were found to be good general and specific combiners, respectively. In conclusion, the stated inbred lines with desirable gca effects and cross combinations with desirable sca effects for grain yield and yield related traits could be used as useful genetic material.

Transcriptomic study of pedicels from GA3-treated table grape genotypes with different susceptibility to berry drop reveals responses elicited in cell wall yield, primary growth and phenylpropanoids synthesis

BackgroundGibberellins (GA3) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA3 is minimal.ResultsCharacterization of responses to GA3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA3 treatment, due to a higher cell wall yield of the isolated fractions of GA3-treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR < 0.05). Gene ontology analysis of upregulated genes showed enrichment in pathways including phenylpropanoids, cell wall metabolism, xylem development, photosynthesis and the cell cycle at 7 days post GA3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis.ConclusionsHigh levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA3, although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.

Cloning and characterization of a homologue of the FLORICAULA/LEAFY gene in Ficus carica L., FcLFY, and its role in flower bud differentiation

Fig (Ficus carica L.) is high in fruit yield and nutritious, and easy to form flower buds. Fig tree, differing from many other fruit trees, has unique flower bud differentiation and fruit-bearing characteristics. Its flower bud (inflorescence) differentiation takes place throughout the shoot elongation period, and it occurs with fruits simultaneously. The entire process of flower bud differentiation to fruit ripening can be completed in one growing season. Studying the unique flowering characteristics of fig provides an important theoretical basis for its cultivation, flowering regulation, fruit production, and molecular breeding. However, the molecular mechanism of flower bud differentiation in fig has not been elucidated, so identifying the relevant genes is essential for understanding the characteristics of fig flower bud differentiation. Here, we isolated a FLORICAULA/LEAFY homologue gene from fig, FcLFY, and identified its function and expression patterns. FcLFY contains a 1377 bp open reading frame and encodes 458 amino acids. The amino acid sequence has a typical LFY/FLO family domain, containing a unique glycine rich region. Moreover, FcLFY expressed the highest in apical bud meristem of fig. In addition, Arabidopsis transgenic lines L23 and L28, which overexpressed FcLFY, bolted 8–9 days and flowered 6 days earlier than WT, while the line C7, which rescued the abnormal flower phenotype of lfy-15 mutant, flowered 8 days earlier than the mutant. Analysis with qRT-PCR further showed that the expressions of flowering promoting genes, including AP1, LFY, CAL and SEP3 were up-regulated while the flowering inhibitory genes FLC and TFL1 were down-regulated in the FcLFY transgenic Arabidopsis. Taken together, FcLFY overexpression shortened the period of vegetative growth, promoted early flowering, partially complemented the phenotypic defects of lfy15 mutant and increased the expression of floral meristem and flower organ genes in Arabidopsis, indicating that FcLFY is involved in the flower bud differentiation and plays an important role in apical meristem formation of fig. This study provides useful information for us to better understand the characterization and function of FcLFY in regulating flower initiation, and is helpful for establishing molecular breeding systems in fig.

Inhibition of B‐cell activation and antibody production by triggering inhibitory signals via the PD‐1/PD‐ligand pathway

The development of donor-reactive antibodies is regarded to be an important barrier limiting long-term outcome of allo- and xenografts. We asked whether enhanced signaling via the co-inhibitory receptor programmed cell death-1 (PD-1; CD279) can downregulate human B-cell activation. Proliferation of human purified CD19(+) B cells was induced by in vitro stimulation with CpG oligodeoxynucleotides (CpG-B). To induce antibody production, peripheral blood mononuclear cells were co-cultured with the porcine B-cell line L23. Triggering of inhibitory signals via the PD-1 receptor was obtained either using a recombinant agonistic soluble ligand (PD-L1.Ig) or L23 transfectants overexpressing membrane-bound human PD-L1 (CD274; L23-PD-L1 cells). Stimulation of purified CD19(+) Bcells with CpG-B resulted in upregulation of PD-1 and strong proliferation. Addition of PD-L1.Ig significantly reduced B-cell proliferation in a dose-dependent manner. A great proportion (~1%) of human circulating Bcells recognizes the epitope galactose-α1,3-galactose-β1,4-N-acetylglucosamine-R (α-gal). Thus, when Bcells-in the presence of Tcell help-were cocultured with α-gal-expressing L23 cells, anti-gal and anti-L23 antibodies could readily be detected in the culture supernatant. The level of induced antibodies was significantly reduced when stimulation was performed by L23-PD-L1 cells. Enhancing inhibitory signals may be part of future protocols to better control humoral immunity to allo- and xenografts.

Naïve Rat NK Cells Control the Onset of T Cell Response

NK cell function in the rat is only defined in a rudimentary way due to missing tools for clear NK cell identification. The present study introduces the congenic LEW.BH-NKC rat strain which allows distinct detection of rat NK cells using commercial antibodies. LEW.BH-NKC rats were exposed in vivo to the porcine B cell line L23 by subcutaneous transfer of L23 cell suspension. We used Luciferase transgeneic L23 cells to follow the course of rejection by living imaging. L23 cells were rejected within five days after placement under the skin thus the rejection is mediated by innate immune responses in the first place. Indeed we found increased percentages of NK cells in the blood, spleen and in draining lymph nodes using flow cytometry methods. Surprisingly, we found as a consequence a decrease in proliferative T cell response in the draining lymph nodes. We identified NK cells as mediators of this regulation by in vitro performed mixed lymphocyte reactions. The remarkable feature was the naive state of NK cells exhibiting the regulative capacity. Furthermore, the regulation was not exclusively mediated by IL-10 as it has been reported before for influence of T cell response by activated NK cells but predominantly by TGF-β. Interestingly, after initiation of the adaptive immune response, NK cells failed to take influence on the proliferation of T cells. We conclude that naive NK cells build up a threshold of activation impulse that T cells have to overcome.

Downregulation of cytolytic activity of human effector cells by transgenic expression of human PD-ligand-1 on porcine target cells

Cellular rejection is a relevant hurdle for successful pig-to-primate xenotransplantation. We have shown previously that the induction of a human anti-pig T cell response (in vitro activation of CD4(+) T cells) can be suppressed by the overexpression of human negative costimulatory ligands (e.g. programmed death receptor ligand, PD-L1) on pig antigen presenting cells. Here, we asked whether PD-L1 mediated enhancement of negative signaling might also be efficient during the effector phase of human anti-pig cellular immune responses. The porcine B-cell line L23 was transfected with human PD-L1, and clones were selected stably expressing PD-L1 with low, medium, or high density. Mock-transfected L23 cells were effectively lysed by human cytotoxic effector cells (IL-2 activated CD8(+) T cells and CD56(+) cells). The lytic potential of the effectors decreased with increasing levels of PD-L1 and was reduced by about 50% in L23-PD-L(high) targets. A proportion of activated CD8(+) effector cells underwent apoptosis when exposed to PD-L1 expressing L23 cells. These data suggest that the overexpression of PD-L1 on target cells may (a) trigger negative signals in effector cells that prevent the release of cytolytic molecules and/or (b) induce apoptosis in the attacking effector cells thereby protecting targets from destruction.

Suppression of Human T-Cell Activation and Expansion of Regulatory T Cells by Pig Cells Overexpressing PD-Ligands

Genetic modification of pigs (e.g., transgenic expression of human complement regulatory molecules or inactivation of alpha1,3galactosyltransferase) enabled the development of promising strategies to overcome hyperacute rejection after pig-to-primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. This report tested the hypothesis that overexpression of human negative costimulatory PD-Ligands (PD-L) in pig antigen presenting cells might be an approach to prevent human anti-pig T-cell responses. The pig B-cell line L23 was transfected with the pIRES-AcGFP vector containing human PD-L1 or PD-L2. Stable transfectants (L23-PD-L1, L23-PD-L2 cells) were established and used for in vitro stimulation of purified human CD4+ T cells. Human CD4+ T cells responded with significantly reduced proliferation to L23-PD-L1 or L23-PD-L2 cells and produced less IL-2, IFNgamma, TNFalpha, IL-4, and IL-5 than cells stimulated with mock-transfected B cells. The concentration of IL-10, however, was increased in CD4+ T cells responding to stimulation with PD-L1 or PD-L2 transfectants. Furthermore, in cultures of CD4+ T cells stimulated for 3 weeks with PD-L1 or PD-L2 transfectants a CD4+CD25(high)Foxp3+ subset showed up that effectively suppressed the activation of conventional CD4+ T cells. These findings imply that PD-1/PD-Ligand pathways are interesting targets to prevent human anti-pig T-cell responses after xenotransplantation, and also suggests that PD-1/PD-Ligand interactions may play a role in the control of the activity and/or homeostasis of regulatory T cells.

Persistence of MHC DR nonexpression on swine cells by introduction of a mutated MHC class II transactivator gene: a comparison with the effect induced by antisense RNA.

CIITA (class II transactivator) is a coactivator essential for transcription of MHC class II genes. In this study, a construct with a mutated CIITA gene with N-terminal domains depleted was constructed. This mutated CIITA (mCIITA) was able to repress DR and DQ expression in 45.0-60.0% of the mCIITA-transfected clones of swine endothelial cell line PIEC and in B cell line L23, as well as in human cell lines HeLa and Raji. Similarly, 30.0-46.7% of swine cell clones containing the human CIITA antisense RNA also failed to express DR molecules. However, the persistence of the DR repression on the cell lines is quite different. Transfection with mCIITA was persistent for at least 120 days, while with the CIITA antisense RNA, persistence existed for only 35-45 days. To explore the underlying mechanism, Raji cells were transfected with pUHD10-3-mCIITA, a mCIITA-containing, doxycycline-dependent plasmid. The intensity of DR repression is correlated quite well with the efficiencies of the mCIITA expression within the cells in a doxycycline dose-dependent manner. This implicates a competition between the mCIITA and its endogenous full-length counterpart. In addition, we were able to show that purified human CD4 T cells did not respond to the mCIITA-transfected PIECs in xenogeneic mixed lymphocyte endothelial reaction (MLER). The stimulating indices (SI) were only 1.0-1.5, compared with 15.2-18.2 for those transfected with empty vector or an initiation codon-depleted mCIITA that is dysfunctional for protein translation. The results we obtained, especially those for persistent suppression of class II genes, show promise for the possible development of mCIITA-transgenic swine for organ/tissue xenotransplantation.

A novel porcine gammaherpesvirus

A novel porcine gammaherpesvirus was detected in the blood of domestic pigs by PCR. With degenerate-primer PCR and subsequent long-distance PCR approaches a 60-kbp genome stretch was amplified. Sequence analysis revealed the presence of the gammaherpesvirus ORFs 03 to 46 as well as a putative chemokine receptor and a v-bcl-2 gene. The 60-kbp sequence was compared with the corresponding sequence of the porcine lymphotropic herpesvirus 1 (PLHV-1) published recently and the sequence of PLHV-2, which was amplified from porcine tonsil. Considerable sequence differences (amino acid identities: 49–89%) were found between the novel virus and PLHV-1 as well as PLHV-2, which were very closely related to each other (amino acid identities: 85–98%). The novel virus had essentially the same genome organization as PLHV-1 and -2 and was therefore designated PLHV-3. Like PLHV-1 and -2, PLHV-3 was frequently found in the blood and in lymphoid organs of domestic and feral pigs from different geographic locations. In the blood, the PLHVs were detected predominantly in B-cells. Indication for latent as well as productive PLHV-3 infection was found in the porcine B-cell line L23. It can be concluded that the PLHVs are widespread and are likely to cause a persistent B-lymphotropic infection. Since PLHV-1 has been implicated in the development of porcine posttransplantation lymphoproliferative disease, all porcine lymphotropic gammaherpesviruses are of concern when pigs are used as donors in xenotransplantation.

Analysis of human CD4 T lymphocyte proliferation induced by porcine lymphoblastoid B cell lines.

This study was undertaken to characterize the two porcine lymphoblastoid cell lines L23 and L35, derived from a pig inoculated by the retrovirus Tsukuba-1, and to determine how they induce a strong human lymphocyte proliferation. Phenotypic characterization was performed by flow cytometry and reverse transcriptase-polymerase chain reaction analyses. Xenogeneic mixed lymphocyte reactions (XMLR) were performed using unfractionated human peripheral blood mononuclear cells (huPBMC) and purified CD4+ T lymphocytes as responding cells, in the presence of blocking antibodies and fusion proteins. The immunoglobulin genes were demonstrated to be rearranged in L23 and L35 cell lines, in agreement with the expression of a B cell phenotype. Both induced a similar proliferation of huPBMCs and purified human CD4+ lymphocytes from adult or cord blood (naïve cells). Proliferation of CD4+ T lymphocytes was completely blocked by anti-SLA-DR plus anti-SLA-DQ mAbs, excluding human lymphocyte transformation by porcine viruses. The frequency of proliferative precursors was inconsistent with that induced by a retroviral superantigen but similar to classical direct xenoantigen presentation as observed with other porcine antigen-presenting cells. Extensive analysis of costimulatory signals led to the identification of the CD28 pathway, in agreement with membrane expression of B7 molecules on L23 and L35 cells, and of the CD2 pathway in L35 cells. These two porcine lymphoblastoid cell lines have been further characterized and clearly identified as belonging to the B cell lineage. By expressing major histocompatibility complex class II antigens and costimulatory molecules, they induce a vigorous proliferative response of human CD4+ lymphocytes through a direct presentation pathway.

Characterization of monoclonal antibodies recognizing immunoglobulin κ and λ chains in pigs by flow cytometry

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igκ) and Ig lambda (Igλ). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igκ and Igλ and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Igλ antiserum and were negative when tested using anti-human Igκ antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Igλ-specific whereas mAb 27.2.1 recognizes the Igκ chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Igλ + B cells in most pigs. In summary, we identified two pan-specific anti-pig Igλ mAbs, one anti-λ mAb that recognizes a λ-light chain subset and one anti-pig Igκ mAb.

Expression level of a transgenic lambda2 chain results in isotype exclusion and commitment to B1 cells.

Two new lambda2 chain-transgenic mouse lines were established, both of which showed stable transgene expression during aging of the mice. The line L23, which expressed the transgene at low levels, exhibited normal B cell development, antibody responses and serum Ig levels. Most of the B cells in this mouse line co-expressed the transgenic lambda2 chain together with an endogenous kappa chain, thus showing poor allelic exclusion of endogenous L chains. On the other hand, high expression of the transgenic lambda2 chain in the other mouse line, L2, resulted in nearly complete exclusion of endogenous L chain isotypes. In this line, the lambda2 transgene was already detectable in the cytoplasm of all preB-II cells and some pro/preB-I cells. Its expression during these early phases obviously inhibited development of conventional B2 cells, since the B cells in the periphery of these mice were almost exclusively of the B1 type. This finding was confirmed by adoptive transfer of transgenic bone marrow into lethally irradiated recipients. Very few B cells were present in the spleen of such recipients. The serum IgM levels of L2 mice were close to normal and the majority of these IgM were associated with the transgenic lambda2 chain. Antibody responses to thymus-dependent antigens in such mice were almost exclusively found to be of IgM class. Together, these findings indicate a developmental bias leading to a predominance of B1 cells in the L2 line.

Suppression of human anti-porcine T-cell immune responses by major histocompatibility complex class II transactivator constructs lacking the amino terminal domain.

The class II transactivator (CIITA) is a bi- or multifunctional domain protein that acts as a transcriptional activator and plays a critical role in the expression of MHC class II genes. We have previously demonstrated that a mutated form of the human CIITA gene, coding for a protein lacking the amino terminal 151 amino acids, acts as a potent dominant-negative suppressor of HLA class II expression. Porcine MHC class II antigens are potent stimulators of direct T-cell recognition by human CD4+ T cells and are, therefore, likely to play an important role in the rejection responses to transgenic pig donors in clinical xenotransplantation. We were, therefore, interested in examining mutated CIITA constructs for their effect on porcine MHC class II expression. Stable transfectants of the porcine vascular endothelial cell line PIEC with mutated CIITA constructs were tested for SLA-DR and SLA-DQ induction by recombinant porcine interferon-gamma. Transient transfectants of the porcine B-cell line L23 with the mutated CIITA constructs were tested for the suppression of constitutive SLA-DR and SLA-DQ expression. T-cell proliferation studies were performed using highly purified human CD4+ T cells. In preliminary studies, we demonstrated that transfection of the PIEC line with full-length human CIITA constructs resulted in strong expression of SLA-DR and SLA-DQ antigens, thus establishing the cross-species effectiveness of human CIITA in the pig. The mutated human CIITA constructs were, therefore, tested in the pig. PIEC clones stably transfected with one of these constructs showed up to 99% suppression of SLA-DR and SLA-DQ antigen induction and marked suppression of SLA-DRA mRNA induction. Moreover, transient transfection of the porcine B-cell line L23 showed up to 90% suppression of constitutive SLA-DR and SLA-DQ antigen expression in 5-8 days. In functional studies, interferon-gamma-stimulated PIEC clones transfected with this mutated CIITA construct failed to stimulate purified human CD4+ T lymphocytes. Mutated human CIITA constructs are potent suppressors of porcine MHC class II expression.