Antibacterial effect of human V gamma 2V delta 2 T cells in vivo

Abstract

V gamma 2V delta 2 cells, a class of T cells found only in primates, are reactive to nonpeptide organophosphate and alkylamine antigens secreted by bacteria and parasites. These cells make up 2-5% percent of human peripheral blood T cells but expand to make up 8-60% of peripheral blood T cells during bacterial and parasitic infections. We show here, using a chimeric severe combined immunodeficiency (SCID) mouse (hu-SCID) model, that human V gamma 2V delta 2 T cells mediate resistance to extracellular gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli and Morganella morganii) bacteria, as assessed by survival, body weight, bacterial loads, and histopathology. Surprisingly, this bacterial resistance was evident 1 day after infection, and bacteria were cleared well before gamma delta T cell expansion was detected 6 days after infection. Decreased resistance in V delta 2 T cell-depleted hu-SCID mice correlated with decreased serum IFN-gamma titers. Intravenous treatment of infected, reconstituted hu-SCID mice with pamidronate, a human V gamma 2V delta 2 T cell-specific aminobisphosphonate antigen, markedly increased the in vivo antibacterial effect of V gamma 2V delta 2 T cells. Therefore, this large pool of antigen-specific, yet immediately reactive memory human V gamma 2V delta 2 T cells is likely to be an important mediator of resistance against extracellular bacterial infection and may bridge the gap between innate and acquired immunity.

Figure 1

Depletion of Vδ2 T cells exacerbated bacterial infection (a–c), whereas live bacterial product augmented the antibacterial effects of Vγ2Vδ2 T cells (d–g). (a) All SCID-beige mice infected with a lethal dose of E. coli (1 × 10⁷ CFUs, administered intraperitoneally) were dead within 2 days, whereas all mice reconstituted with human PBMCs survived this lethal infection. (b) Six of ten SCID-beige mice reconstituted with human PBMCs depleted of Vδ2 T cells died of E. coli infection (3 × 10⁷ CFUs, administered intraperitoneally); in contrast, only one of ten mice receiving mock-depleted PBMCs died. (c) Reconstitution of SCID mice with intraperitoneal PBMCs depleted of Vδ2 T cells resulted in higher bacterial loads in the peritoneal lavages of these mice 5 days after intraperitoneal inoculation of M. morganii (3 × 10⁷ CFUs). (d) SCID mice (n = 5 for each group) reconstituted intraperitoneally with PBMCs pretreated with IBA, a natural Vγ2Vδ2 T cell–specific antigen secreted by bacteria, had lower numbers of bacterial CFUs in the peritoneal lavage (e) less loss of body weight (*P < 0.05), and (f) markedly less liver degeneration than those reconstituted intraperitoneally with medium-pretreated PBMCs 5 days after intraperitoneal infection with 3 × 10⁷ CFUs of M. morganii (×200, hematoxylin and eosin staining). Bottom panel: IBA group. Top panel: medium control. (g) SCID mice reconstituted intraperitoneally with IBA-pretreated PBMCs (IBA) had lower numbers of bacterial CFUs than those receiving medium-pretreated PBMCs (Medium) 5 days after intravenous infection with 3 × 10⁷ CFUs of M. morganii.

Figure 2

Treatment with aminobisphosphonate antigen but not bisphosphonate in vivo enhanced the antibacterial effect of PBMCs (a–c); human Vγ2Vδ2 T cells expanded in SCID mice (d, e). SCID mice intraperitoneally reconstituted with PBMCs and treated with intravenous pamidronate (10 mg/kg body weight), a Vγ2Vδ2 T cell–specific aminobisphosphonate antigen, 2 hours after infection with intravenous M. morganii (3 × 10⁷ CFUs), had fewer liver and spleen CFUs than did reconstituted, infected SCID mice treated with etidronate (10 mg/kg body weight), an antigenically inactive bisphosphonate analogue of pamidronate. (a) Four days after infection, SCID mice (n = 5) reconstituted intraperitoneally with PBMCs pretreated with pamidronate had lower numbers of CFUs than those reconstituted with etidronate-pretreated PBMCs 27 hours after intraperitoneal infection with E. coli (5 × 10⁶ CFUs) (b) or 17 hours after infection with S. aureus (2 × 10⁶ CFUs) (c); n = 5 for each group. (d) Flow cytometry profile, and (e) absolute numbers of Vγ2Vδ2 T cells in the peritoneal lavage: Human Vγ2Vδ2 T cells in the peritoneal lavage of SCID mice (n = 5 in each group) reconstituted intraperitoneally with IBA-pretreated PBMCs were expanded by day 7 after intraperitoneal infection with 3 × 10⁷ CFUs of M. morganii (IBA + M. morganii), compared with peritoneal lavage Vγ2Vδ2 T cells from reconstituted SCID mice that were mock-infected (IBA). Each SCID mouse was reconstituted with PBMCs that contained 2.5 × 10⁵ γδ T cells. Data were representative of three experiments.

Figure 3

Vδ2 T cells and IFN-γ play a crucial role in monocyte-mediated killing of extracellular bacteria. IBA-pretreated PBMCs exposed in vitro to dead bacteria for 17 hours induced γδ T cell–dependent IFN-γ secretion (a). At 17 hours after intraperitoneal infection with E. coli (5 × 10⁶ CFUs), SCID mice (n = 5 in each group) reconstituted intraperitoneally with IBA-pretreated, mock-depleted PBMCs had higher levels of serum human IFN-γ than did mice reconstituted with IBA-pretreated PBMCs depleted of Vδ2 T cells (b). Intracellular IFN-γ was produced by 9.7% of γδ T cells harvested from the peritoneal lavage of SCID mice that were reconstituted intraperitoneally with pamidronate-pretreated PBMCs (or IBA-pretreated PBMCs, data not shown) and infected intraperitoneally with E. coli (5 × 10⁶ CFUs) (c, bottom), while only 1.6% of γδ T cells harvested from reconstituted and mock-infected SCID mice produced IFN-γ (c, top). Monocytes from PBMC cultures that were depleted of Vδ2 T cells killed less than half as many bacteria than did monocytes from mock-depleted cultures. Inclusion of neutralizing mAbs to IFN-γ in the culture abrogated the γδ T cell–dependent monocyte-mediated killing of M. morganii. Addition of IFN-γ to the Vδ2 T cell–depleted culture completely reconstituted the antibacterial effect lost by depletion of Vδ2 T cells (d). Use of pamidronate-pretreated PBMCs resulted in data similar to that obtained using IBA-pretreated PBMCs (data not shown). Data were representative of two to four experiments.