BTN3A Targeting Vγ9Vδ2 T Cells Antimicrobial Activity Against Coxiella burnetii-Infected Cells

Abstract

Vγ9Vδ2 T cells have been reported to participate to the immune response against infectious diseases such as the Q fever caused by Coxiella burnetii infection. Indeed, the number and proportion of Vγ9Vδ2 T cells are increased during the acute phase of Q fever. Human Vγ9Vδ2 T cell responses are triggered by phosphoantigens (pAgs) produced by pathogens and malignant cells, that are sensed via the membrane receptors butyrophilin-3A1 (BTN3A1) and -2A1 (BTN2A1). Here, by using CRISPR-Cas9 inactivation in THP-1 cells, we show that BTN3A and BTN2A are required to Vγ9Vδ2 T cell response to C. burnetii infection, though not directly involved in the infection process. Furthermore, C. burnetii-infected monocytes display increased BTN3A and BTN2A expression and induce Vγ9Vδ2 T cell activation that can be inhibited by specific antagonist mAb. More importantly, we show that the antimicrobial functions of Vγ9Vδ2 T cells towards C. burnetii are enhanced in the presence of an BTN3A activating antibody. This supports the role of Vγ9Vδ2 T cells in the control of C. burnetii infection and argues in favor of targeting these cells as an alternative treatment strategy for infectious diseases caused by intracellular bacteria.

Keywords: Coxiella burnetii; Vγ9Vδ2 T cells; antimicrobial immunity; butyrophilin; therapeutic approaches.

Figure 1

Bacterial infections modulate BTN3A and BTN2A expression. Monocytes isolated from healthy donors (n = 4) were infected with C. burnetii strains (50 MOI) or with M. tuberculosis (5 MOI) for 24 hours. (A) The relative gene expression of BTN3A isoforms (A1, A2, A3) and (B) the BTN3A protein expression were investigated by qRT-PCR and flow cytometry, respectively. (C) The relative gene expression of BTN2A isoforms (A1, A2) and (D) the BTN2A protein expression were investigated by qRT-PCR and flow cytometry, respectively. Data were analyzed using a normality test and a parametric t test. Values represent mean ± standard deviation. *p < 0.05, **p < 0.01, and ****p < 0.0001.

Figure 2

Involvement of BTN3A and BTN2A in C. burnetii infection. CRISPR-Cas9-mediated inactivation of BTN3A or BTN2A was performed in THP-1 cell line. THP-1 cells transduced with a guide targeting all BTN3A isoforms (BTN3KO) or all BTN2A isoforms (BTN2AKO) or with an irrelevant CRISPR guide (mock) for control cells were infected with C. burnetii NM1 (50 MOI) (n = 3). (A) After 4 and 24 hours of infection, the number of bacterial DNA copies within THP-1 cells was assessed by qPCR. (B) THP-1 cells were incubated with C. burnetii for 4 h (day 0), then washed to eliminate free bacteria and incubated for 4 days. Each day, the number of bacterial DNA copies was evaluated by qPCR. Values represent mean ± standard deviation.

Figure 3

Involvement of BTN3A and BTN2A in the inflammatory response to C. burnetii infection. THP-1 cells transduced with an irrelevant CRISPR guide (mock) or a guide targeting all BTN2A isoforms (BTN2AKO) or all BTN3A isoforms (BTN3AKO) were infected with C. burnetii NM1 (100 MOI) (n = 3). After 24 hours infection, the expression of genes involved in the inflammatory (TNF, IL1B, IL6, IFNG, CXCL10) or immunoregulatory (IL10, TGFB1, IL1RA, CD163) response was investigated by quantitative reverse-transcription polymerase chain reaction after normalization with housekeeping actin gene as endogenous control. Data are illustrated as (A) hierarchical clustering obtained using ClustVis webtool or (B) relative quantity of investigated genes. (C) After 24 hours infection, TNF-α, IL-1β, IFN-γ, IL-6, IL-10, and TGF-β release were evaluated in the culture supernatants by ELISA assay. Data were analyzed using a normality test and a parametric t test. Values represent mean ± standard error. *p < 0.05 and **p < 0.01.

Figure 4

Infection with C. burnetii leads BTN3A and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. (A) Monocytes isolated from healthy donors (n = 3) previously infected 24 hours with C. burnetii NM1 (50 or 100 MOI) or with M. tuberculosis (1, 5 or 10 MOI) were co-cultured with autologous Vγ9Vδ2 T cells (E:T ratio of 1:1). Vγ9Vδ2 T cell degranulation (%CD107ab+ cells) was assessed after 4 hours of co-culture by flow cytometry. (B–D) Monocytes isolated from healthy donors (n = 4) previously infected 24 hours with (C) burnetii strains (50 MOI) or with M. tuberculosis (5 MOI) were co-cultured with Vγ9Vδ2 T cells expanded from healthy donor (E:T ratio of 1:1) in the presence of (B) anti-BTN2A (clone 7.48), (C) anti-BTN3A (clone 103.2) or (D) anti-BTN3A (clone 20.1) antibodies (10µg/ml). Vγ9Vδ2 T cell degranulation (%CD107ab+ cells) was assessed after 4 hours of co-culture by flow cytometry. (E) The cytotoxicity was assessed by flow cytometry as the percentage of Caspase 3/7⁺ cells in the target cell population after 4 hours of co-culture in presence of anti-BTN3A antibody (clone 20.1) (10µg/ml). Data were analyzed using a normality test and a parametric t test. Values represent mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 5

Anti-BTN3A agonist antibody increases antimicrobial activity of Vγ9Vδ2 T cells towards C. burnetii infected monocytes. (A, B) Monocytes isolated from healthy donors (n = 4) previously infected 24 hours with C. burnetii NM1 (50 MOI) were co-cultured with autologous Vγ9Vδ2 T cells (E:T ratio of 1:1) in the presence of anti-BTN3A antibody (clone 20.1) (0-10 µg/ml). After 4 hours of co-culture, C. burnetii load was measured by (A) flow cytometry and (B) qPCR. Data were analyzed using a normality test and a parametric t test. Values represent mean ± standard deviation. *p < 0.05, **p < 0.01.

Figure 6

Anti-BTN3A agonist antibody increases the secretion of cytokines and cytotoxic molecules in Vγ9Vδ2 T cell/infected-monocyte co-cultures. Monocytes isolated from healthy donors (n = 4) previously infected 24 hours with C. burnetii NM1 (50 MOI) or with M. tuberculosis (5 MOI) were co-cultured with autologous Vγ9Vδ2 T cells (E:T ratio of 1:1) in the presence of anti-BTN3A antibody (clone 20.1) (0-10 µg/ml). After 4 hours of co-culture, the culture supernatants were analyzed for the presence of cytokines (A, left panel) and cytotoxic molecules (B, right panel) by ELISA assay. Data were analyzed using a normality test and a parametric t test. Values represent mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.