College of Agriculture & Natural Resources
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Item DIFFERENTIATION AND REGULATION OF BOVINE TH2 CELLS(2024) Kandel, Anmol; Xiao, Zhengguo Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Memory CD4+ T cells, specifically type-2 (Th2) cells, are pivotal in defending against infections caused by extracellular pathogens, including several economically important parasites. However, whether interleukin-4 (IL4) expression is a signature feature of bovine Th2 cells likewise in mice and humans is unclear. Pasture-raised cattle, routinely exposed to extracellular parasites such as Ostertagia ostertagi (OO), are likely to develop a typical Th2 memory response. Therefore, using cytokine induction assay, we evaluated the circulatory memory bovine T cell profile of these cattle and also analyzed if the expression of presumptuous memory marker, CD45RO, is reliable in identifying memory bovine T cells. Surprisingly, the majority of the memory CD4+ T cells dominantly produced interferon-gamma (IFNγ), with only a small fraction co-expressing IL4, and memory bovine T cell identification did not correlate with CD45RO expression. Results suggested that cattle naturally exposed to extracellular parasites do not develop typically IL4 dominant Th2 response. To further investigate these results, resting CD4+ T cells isolated from healthy cattle blood were cultured under simple in vitro Th2 culture. Analysis of differentiated cells through flow cytometry revealed limited IL4 protein detection, which was in line with the lack of upregulation of IL4 and its master regulator GATA3 transcripts shown by the quantitative polymerase chain reaction (qPCR) assay. To validate whether differentiated cells were actually Th2, unbiased proteomic analysis was conducted. Based on differentially expressed 397 proteins between differentiated cells and naïve phenotype, bovine Th2 differentiation was validated; nonetheless, the process was not found to be associated with IL4 induction. Moreover, despite using published strategies from mice and humans, such as reducing T cell receptor (TCR) stimulation strength and adding exogenous recombinant bovine IL4, the expression of IL4 could not be significantly enhanced. Interestingly, differentiated bovine Th2 cells proliferated in the presence of OO antigens, suggesting that extracellular parasites could influence bovine Th2 differentiation, at least in vitro. To validate the results from pathogen-infected tissues and in vitro culture, a panel of anti-parasitic CD4+ single T cell clones was established from five pasture-raised cattle that were infected with OO. Evaluation of memory responses exhibited by the anti-parasitic CD4+ single T cell clones strongly supported IFNγ dominant memory response, and only 20% of them co-expressed IL4 through a small subset of IFN γ + cells. All the data pointed out that bovine CD4+ T cell differentiation is partially distinct from those in mice and humans, and IL4 expression is not a hallmark feature of the bovine Th2 cells.Item Allele-Specific Expression of CD4+ T Cells in Response to Marek’s Disease Virus Infection(MDPI, 2019-09-17) Bai, Hao; He, Yanghua; Ding, Yi; Carrillo, José A.; Selvaraj, Ramesh K.; Zhang, Huanmin; Chen, Jilan; Song, JiuzhouMarek’s disease (MD) is a T cell lymphoma disease induced by Marek’s disease virus (MDV), a highly oncogenic α herpesvirus primarily affecting chickens. MD is a chronic infectious disease that threatens the poultry industry. However, the mechanisms of genetic resistance for MD are complex and not completely understood. In this study, to identify high-confidence candidate genes of MD genetic resistance, high throughput sequencing (RNA-seq) was used to obtain transcriptomic data of CD4+ T cells isolated from MDV-infected and non-infected groups of two reciprocal crosses of individuals mating by two highly inbred chicken lines (63 MD-resistant and 72 MD-susceptible). After RNA-seq analysis with two biological replicates in each group, we identified 61 and 123 single nucleotide polymorphisms (SNPs) (false discovery rate (FDR) < 0.05) annotated in 39 and 132 genes in intercrosses 63 × 72 and 72 × 63, respectively, which exhibited allele-specific expression (ASE) in response to MDV infection. Similarly, we identified 62 and 79 SNPs annotated in 66 and 96 genes in infected and non-infected groups, respectively. We identified 534 and 1543 differentially expressed genes (DEGs) (FDR < 0.05) related to MDV infection in intercrosses 63 × 72 and 72 × 63, respectively. We also identified 328 and 20 DEGs in infected and non-infected groups, respectively. The qRT-PCR using seven DEGs further verified our results of RNA-seq analysis. The qRT-PCR of 11 important ASE genes was performed for gene functional validation in CD4+ T cells and tumors. Combining the analyses, six genes (MCL1, SLC43A2, PDE3B, ADAM33, BLB1, and DMB2), especially MCL1, were highlighted as the candidate genes with the potential to be involved in MDV infection. Gene-set enrichment analysis revealed that many ASE genes are linked to T cell activation, T cell receptor (TCR), B cell receptor (BCR), ERK/MAPK, and PI3K/AKT-mTOR signaling pathways, which play potentially important roles in MDV infection. Our approach underlines the importance of comprehensive functional studies for gaining valuable biological insight into the genetic factors behind MD and other complex traits, and our findings provide additional insights into the mechanisms of MD and disease resistance breeding in poultry.Item Synergistic Activation of Bovine CD4+ T Cells by Neutrophils and IL-12(MDPI, 2021-06-03) Xiao, Zhengguo; Kandel, Anmol; Li, LeiCD4+ T cell activation requires inflammatory cytokines to provide a third signal (3SI), such as interleukin-12 (IL-12). We recently reported that bovine neutrophils can enhance the activation of bovine CD4+ T cells. To explore the interactions between neutrophils and third signal cytokines in bovine CD4+ T cell activation, naïve CD4+ T cells were isolated from cattle lymph nodes and stimulated for 3.5 days with anti-bovine CD3 (first signal; 1SI), anti-bovine CD28 (second signal; 2SI), and recombinant human IL-12 (3SI) in the presence or absence of neutrophils harvested from the same animals. Indeed, the strongest activation was achieved in the presence of all three signals, as demonstrated by CD25 upregulation, IFNγ production in CD4+ T cells, and secretion of IFNγ and IL-2 in cell supernatants. More importantly, 1SI plus neutrophils led to enhanced CD25 expression that was further increased by IL-12, suggesting synergistic action by IL-12 and neutrophils. Consistently, neutrophils significantly increased IFNγ production in 1SI plus IL-12-stimulated CD4+ T cells. Our data suggest the synergy of neutrophils and IL-12 as a novel regulator on bovine CD4+ T cell activation in addition to three signals. This knowledge could assist the development of immune interventions for the control of infectious diseases in cattle.Item Differential Expression of CD45RO and CD45RA in Bovine T Cells(MDPI, 2022-06-04) Kandel, Anmol; Li, Lei; Hada, Akanksha; Xiao, ZhengguoEffective vaccination induces immune memory to protect animals upon pathogen re-encounter. Despite contradictory reports, bovine memory T cells are identified based on two isoforms of CD45, expression of CD45RO plus exclusion of CD45RA. In this report, we contrasted CD45RA/RO expression on circulatory T cells with IFNγ and IL4 expression induced by a conventional method. To our surprise, 20% of cattle from an enclosed herd did not express CD45RO on T cells without any significant difference on CD45RA expression and IFNγ or IL4 induction. In CD45RO expressing cattle, CD45RA and CD45RO expressions excluded each other, with dominant CD45RO (>90%) expression on gamma delta (γδ) followed by CD4+ (60%) but significantly higher CD45RA expression on CD8+ T cells (about 80%). Importantly, more than 80% of CD45RO expressing CD4+ and CD8+ T cells failed to produce IFNγ and IL-4; however, within the cytokine inducing cells, CD4+ T cells highly expressed CD45RO but those within CD8+ T cells mostly expressed CD45RA. Hence, CD45RO is not ubiquitously expressed in cattle, and rather than with memory phenotype, CD45RA/RO expression are more associated with distinct T cell subtypes.