Evidence of innate training in bovine γδ T cells following subcutaneous BCG administration

Abstract

The Bacillus Calmette Guerin (BCG) vaccine has been shown to induce non-specific protection against diseases other than tuberculosis in vaccinated individuals, attributed to the induction of trained immunity. We have previously demonstrated that BCG administration induces innate immune training in mixed peripheral blood mononuclear cells and monocytes in calves. Gamma Delta (γδ) T cells are non-conventional T cells that exhibit innate and adaptive immune system features. They are in higher proportion in the peripheral blood of cattle than humans or rodents and play an essential role in bovine immune response to pathogens. In the current study, we determined if BCG administration induced innate immune training in bovine γδ T cells. A group of 16 pre-weaned Holstein calves (2-4 d age) were enrolled in the study and randomly assigned to vaccine and control groups (n=8/group). The vaccine group received two doses of 10⁶ colony forming units (CFU) BCG Danish strain subcutaneously, separated by 2 weeks. The control group remained unvaccinated. Gamma delta T cells were purified from peripheral blood using magnetic cell sorting three weeks after receiving the 1^st BCG dose. We observed functional changes in the γδ T cells from BCG-treated calves shown by increased IL-6 and TNF-α cytokine production in response to in vitro stimulation with Escherichia coli LPS and PAM3CSK4. ATAC-Seq analysis of 78,278 regions of open chromatin (peaks) revealed that γδ T cells from BCG-treated calves had an altered epigenetic status compared to cells from the control calves. Differentially accessible peaks (DAP) found near the promoters of innate immunity-related genes like Siglec14, Irf4, Ifna2, Lrrfip1, and Tnfrsf10d were 1 to 4-fold more accessible in cells from BCG-treated calves. MOTIF enrichment analysis of the sequences within DAPs, which explores transcription factor binding motifs (TFBM) upstream of regulatory elements, revealed TFBM for Eomes and IRF-5 were among the most enriched transcription factors. GO enrichment analysis of genes proximal to the DAPs showed enrichment of pathways such as regulation of IL-2 production, T-cell receptor signaling pathway, and other immune regulatory pathways. In conclusion, our study shows that subcutaneous BCG administration in pre-weaned calves can induce innate immune memory in the form of trained immunity in γδ T cells. This memory is associated with increased chromatin accessibility of innate immune response-related genes, thereby inducing a functional trained immune response evidenced by increased IL-6 and TNF-α cytokine production.

Keywords: BCG vaccine; bovine γδ T cells; chromatin accessibility; epigenetic reprogramming; trained immunity.

Figure 1

Altered in vitro cytokine responses in bovine γδ T cells following BCG immunization. 10⁶ CFU of BCG was administered subcutaneously to preweaned calves in 2 doses at a 2-week interval. The control group remained unvaccinated. Peripheral blood was collected 1 week after the 2^nd BCG dose, and γδ T cells were separated by MACS purification. γδ T cells were stimulated in vitro with either mock, LPS, or PAM3CSK for 72 hours. Cell culture supernatants were collected, and ELISA was performed to measure innate cytokine production. Data represented as mean ± SEM. P-value as determined by 2-way ANOVA with Sidak’s multiple comparisons test.

Figure 2

Differential chromatin accessibility in bovine γδ T cells from BCG-immunized calves. ATAC sequencing was performed on γδ T cells from BCG and control calves collected 2 weeks after the 2^nd BCG dose, using the Tn5 transposase. Next-gen sequencing was performed on the DNA libraries. After filtering out low-quality reads, peak calling was performed, followed by differential accessible peak analysis of the BCG and control group. The volcano plot shows the results from the DAP analysis with FDR <0.1%.

Figure 3

Relative gene expression of select genes proximal to the DAPs. The DAPs were annotated to known genomic features. A few of these genes were selected, and quantitative PCR was performed after in vitro stimulation with either mock, LPS, PAM3CSK, or Poly IC/Imiquimod for 4 hours. The CT values were normalized with RPS9, and relative gene expression was determined by the 2^-ΔΔCT method. The P-values were obtained by performing an unpaired t-test on the ΔCT values.