Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

Characterization of Bovine Intraepithelial T Lymphocytes in the Gut.

Intraepithelial T lymphocytes (T-IELs), which constitute over 50% of the total T lymphocytes in the animal, patrol the mucosal epithelial lining to defend against pathogen invasion while maintaining gut homeostasis. In addition to expressing T cell markers such as CD4 and CD8, T-IELs display T cell receptors (TCR), including either TCRαß or TCRγδ. Both humans and mice share similar T-IEL subsets: TCRγδ+, TCRαß+CD8αα+, TCRαß+CD4+, and TCRαß+CD8αß+. Among these subsets, human T-IELs are predominantly TCRαß+ (over 80%), whereas those in mice are mostly TCRγδ+ (~60%). Of note, the majority of the TCRγδ+ subset expresses CD8αα in both species. Although T-IELs have been extensively studied in humans and mice, their profiles in cattle have not been well examined. Our study is the first to characterize bovine T-IELs using flow cytometry, where we identified several distinct features. The percentage of TCRγδ+ was comparable to that of TCRαß+ T-IELs (both ~50% of CD3+), and the majority of bovine TCRγδ+ T-IELs did not express CD8 (CD8-) (above 60%). Furthermore, about 20% of TCRαß+ T-IELs were CD4+CD8αß+, and the remaining TCRαß+ T-IELs were evenly distributed between CD4+ and CD8αß+ (~40% of TCRαß+ T-IELs each) with no TCRαß+CD8αα+ identified. Despite these unique properties, bovine T-IELs, similar to those in humans and mice, expressed a high level of CD69, an activation and tissue-retention marker, and a low level of CD62L, a lymphoid adhesion marker. Moreover, bovine T-IELs produced low levels of inflammatory cytokines such as IFNγ and IL17A, and secreted small amounts of the immune regulatory cytokine TGFß1. Hence, bovine T-IELs' composition largely differs from that of human and mouse, with the dominance of the CD8- population among TCRγδ+ T-IELs, the substantial presence of TCRαß+CD4+CD8αß+ cells, and the absence of TCRαß+CD8αα+ T-IELs. These results provide the groundwork for conducting future studies to examine how bovine T-IELs respond to intestinal pathogens and maintain the integrity of the gut epithelial barrier in animals.

Differential Expression of CD45RO and CD45RA in Bovine T Cells.

Effective 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.

Synergistic Activation of Bovine CD4+ T Cells by Neutrophils and IL-12.

CD4+ 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.