Antibacterial effect of human Vγ2Vδ2 T cells in vivo

Vγ2Vδ2 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 Vγ2Vδ2 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 γδ T cell expansion was detected 6 days after infection. Decreased resistance in Vδ2 T cell–depleted hu-SCID mice correlated with decreased serum IFN-γ titers. Intravenous treatment of infected, reconstituted hu-SCID mice with pamidronate, a human Vγ2Vδ2 T cell–specific aminobisphosphonate antigen, markedly increased the in vivo antibacterial effect of Vγ2Vδ2 T cells. Therefore, this large pool of antigen-specific, yet immediately reactive memory human Vγ2Vδ2 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.

Studies in mice suggest that γδ T cells have diverse functions, mediating host defense in preventing lethal herpes simplex type 1–induced encephalitis (1) and providing protective immunity against malaria (2) and antibacterial effects in tuberculosis (3) and listeriosis (4). γδ T cells also play a role in immunoregulation by ameliorating the severity of adjuvant arthritis (5), and enhancing immune tolerance in several model systems (6–9).

Several lines of evidence also implicate γδ T cells as being important in human bacterial infections. Polyclonal Vγ2Vδ2 T cells expand in vivo, typically to comprise 8–60% of all peripheral blood T cells during a variety of bacterial diseases such as tuberculosis (mean, 14%) (10), salmonellosis (mean, 18%) (11), tularemia (mean, 31%) (12), brucellosis (mean, 29%) (13), listeriosis (mean, 12%) (14), and ehrlichiosis (mean, 57%) (15). However, there is no direct in vivo evidence showing antibacterial effects of human Vγ2Vδ2 T cells.

Found only in primates, human Vγ2Vδ2 T cells recognize, in a T cell receptor–dependent, MHC- and CD1-unrestricted manner, nonpeptide alkylamine and organophosphate antigens secreted by bacteria (16–18). The human pathogens Morganella morganii (18), Escherichia coli, Yersinia enterocolitica (19, 20), Salmonella typhimurium (11, 21), and Listeria monocytogenes (22, 23) produce the antigenic alkylamines isobutylamine (IBA; S. typhimurium, Y. enterocolitica, E. coli, M. morganii), isoamylamine, and n-butylamine (L. monocytogenes), and these bacterial infections cause in vivo or in vitro γδ T cell expansions in humans. In contrast to human γδ T cells, murine γδ T cells do not respond to nonpeptide alkylamine or organophosphate antigens; therefore, it is not at all certain that the biological functions of mouse γδ T cells are the same as those in primates. Furthermore, the in vivo functions in humans of these alkylamines and organophosphate antigens are unknown. Here, using a chimeric SCID mouse model, we provide evidence that human Vγ2Vδ2 T cells are important mediators of natural immunity against extracellular bacterial infection. Furthermore, treatment or prophylaxis of human bacterial infections with Vγ2Vδ2-specific nonpeptide antigens either in vitro or in vivo can markedly enhance γδ T cell–mediated antibacterial effects.

Depletion of Vδ2 T cells exacerbates bacterial infection. To determine whether human γδ T cells mediate antibacterial effects in vivo, we reconstituted SCID mice with human PBMCs that were either mock-depleted or depleted of γδ T cells, and challenged the mice with E. coli or M. morganii, two gram-negative bacteria that are important causes of urinary tract infections and urosepsis. SCID mice receiving a lethal dose of E. coli (1 × 10⁷ CFUs) alone all died of infection within 2 days, whereas SCID mice receiving a lethal dose of bacteria and human PBMCs all survived (Figure 1a). These data suggest that human PBMCs play a crucial role in protecting against bacterial infection, and that residual mouse immune cells have negligible effects. This hu-SCID infection model enables us to study further the antibacterial function of human Vγ2Vδ2 T cells. Six of ten SCID mice reconstituted with human PBMCs depleted of Vδ2 T cells died of infection, whereas only one of ten SCID mice reconstituted with human PBMCs that had been mock-depleted of Vδ2 T cells died (P = 0.0307, Figure 1b). In addition, in contrast to the SCID mice reconstituted with mock-depleted human PBMCs, SCID mice reconstituted with human PBMCs depleted of Vδ2 T cells had up to a 2 log₁₀ increase in bacterial CFUs in the peritoneal lavage 5 days after infection (Figure 1c). These results strongly suggest that human Vγ2Vδ2 T cells mediate antibacterial activity against E. coli and M. morganii.

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.

Antibacterial effects of Vγ2Vδ2 T cells are enhanced by live bacterial products. Bacteria such as M. morganii (18), E. coli (21), Y. enterocolitica (21), S. typhimurium (11, 21), and L. monocytogenes (22, 23) stimulate Vγ2Vδ2 T cell expansion, and secrete IBA and other alkylamine antigens at concentrations up to 4 mM, whereas dead bacteria do not produce IBA (18). IBA is recognized in vitro by human Vγ2Vδ2 T cells in a TCR-dependent, MHC- and CD1-unrestricted manner (18). However, the in vivo biological functions of alkylamine antigens are unknown. We speculated that these alkylamine antigens, which are secreted by live bacteria and cannot be recognized by mouse γδ T cells, regulate the antibacterial effects of human Vγ2Vδ2 T cells in vivo.

To determine whether stimulation of PBMCs with the live bacterial product IBA could enhance the antibacterial effect of PBMCs in vivo, we reconstituted SCID mice with IBA-pretreated (1 mM) PBMCs that had been washed prior to inoculation. SCID mice reconstituted with IBA-pretreated PBMCs had a lower number of bacterial CFUs (nearly 2 log₁₀ fewer), lost less body weight, and had milder hepatocyte degeneration than those reconstituted with mock-pretreated PBMCs (Figure 1, d–f). In vitro, human Vγ2Vδ2 T cells expanded and produced cytokines in response to stimulation only with IBA, not with dead bacteria and LPS (our unpublished observations).

IBA did not activate αβ T cells, NK cells, NK T cells, or monocytes, and did not show toxicity at concentrations of less than 2 mM (data not shown). Thus, the live bacterial product IBA specifically activated Vγ2Vδ2 T cells, which enhanced the antibacterial activity of human PBMCs in vivo.

Intraperitoneal inoculation with both PBMCs and M. morganii might be regarded as creating only local and not systemic antibacterial effects. We therefore reconstituted mice with PBMCs intraperitoneally and inoculated with M. morganii intravenously. SCID mice receiving IBA-pretreated PBMCs had up to tenfold fewer bacterial CFUs than those receiving medium-pretreated PBMCs (Figure 1g), indicating that the PBMCs were mediating a systemic antibacterial effect. Thus, in this model, human Vγ2Vδ2 T cells play a very important role against bacterial infection, and this effect is augmented in vivo by the live bacterial product IBA.

Treatment with Vγ2Vδ2 T cell–specific antigens in vivo enhanced the antibacterial effect of PBMCs. Since the natural Vγ2Vδ2 T cell–specific antigen IBA enhanced the antibacterial effect of PBMCs in vivo, we speculated that pharmaceutical Vγ2Vδ2 T cell–specific antigens (28, 29) such as pamidronate, alendronate, or risedronate, may have similar effects. Like IBA, these potent aminobisphosphonate antigens stimulate Vγ2Vδ2 T cells in a TCR-dependent, MHC- and CD1-unrestricted manner (30, 31), and have a proven track record of safety (32, 33).

We reconstituted SCID mice intraperitoneally with untreated PBMCs, and infected the mice intravenously with M. morganii. Two hours after infection, when the mice were clinically ill, pamidronate was injected either intravenously (Figure 2a) or intraperitoneally (data not shown), and the mice were sacrificed on day 4 to quantitate bacterial CFUs in their organs. Reconstituted SCID mice treated with the antigen pamidronate appeared much healthier and had up to 2.5 log₁₀ fewer bacterial CFUs in their spleens than those treated with the antigenically inactive bisphosphonate etidronate (Figure 2a). We also treated the reconstituted SCID mice with pamidronate 2 hours prior to M. morganii infection, and produced similar results (data not shown). Neither pamidronate nor etidronate had direct antibacterial effects in vitro, and neither drug was toxic to cultured human PBMCs at a concentration fivefold higher than that used in this study. Thus, treatment of reconstituted SCID mice that were clinically ill from M. morganii infection, with an antigen specific for Vγ2Vδ2 T cells, had a remarkable systemic antibacterial effect.

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.

Previous studies have demonstrated that human γδ T cells are expanded in vivo and in vitro by a variety of gram-positive and gram-negative bacterial infections (reviewed in ref. 34), prompting us to investigate the protective role of human γδ T cells against other bacteria. Pretreatment of PBMCs with pamidronate rendered these PBMCs up to 100-fold more effective in mediating antibacterial effects in reconstituted SCID mice infected with gram-negative E. coli (Figure 2b) or gram-positive S. aureus (Figure 2c), than infected SCID mice reconstituted with PBMCs pretreated with etidronate. Taken together, these data strongly suggest that pamidronate and other Vγ2Vδ2 T cell antigens might be useful as prophylaxis or treatment in vivo for both gram-positive and gram-negative extracellular bacterial infection.

Expansion of human γδ T cells in SCID mice. To examine the fate of T cells in this in vivo model, SCID mice reconstituted with IBA-pretreated PBMCs were mock-infected or infected with M. morganii, and then sacrificed on day 7 for determination of the number of peritoneal γδ T cells by flow cytometry. γδ T cells had not expanded in the peritoneal cavity by days 3–4 after infection (data not shown). By 7 days after infection, human Vγ2Vδ2 T cells in the peritoneal lavage of the infected hu-SCID mice comprised 72% of CD3⁺ T cells (Figure 2d). There were an average of 7.2 × 10⁵ human γδ T cells in the peritoneal cavities of the infected mice, compared with 2.9 × 10⁵ cells in the uninfected mice (Figure 2, d and e). Human Vγ2Vδ2 T cells in the blood of the infected hu-SCID mice comprised 19.42% of CD3⁺ T cells, compared with 7.3% in the blood of the mock-infected mice. The Vγ2Vδ2 T cells were also detectable from other organs of infected SCID mice, such as liver, spleen, and bone marrow, by 7 days after infection. In contrast, it was difficult to find these cells in the organs of uninfected mice. Since the SCID mice received PBMCs that contained approximately 2.5 × 10⁵ γδ T cells (approximately 2% of CD3⁺ T cells), we estimate that there was an increase of less than 2.5-fold in absolute numbers of human γδ T cells in infected mice by 7 days after infection, compared with uninfected mice. By 12 days after infection, most human PBMCs had migrated to the mouse spleen. There was a mean total of 13.4 × 10⁶ human Vγ2Vδ2 T cells found in the spleen of each infected hu-SCID mouse, whereas a mean of only 1.8 × 10⁶ γδ T cells were found in the spleens of mock-infected hu-SCID mice. Similar results were observed in expansion of Vγ2Vδ2 T cells when mice were intravenously reconstituted with PBMCs and intraperitoneally infected with M. morganii, or vice versa, or both were inoculated intraperitoneally. Thus, bacterial infection resulted in the expansion of γδ T cells in this in vivo hu-SCID mouse model, reflecting the expansion seen in peripheral blood during infection in man.

IFN-γ production is partially dependent on Vδ2 T cells. Exposure of Vγ2Vδ2 T cells to bacteria or nonpeptide bacterial antigens results in secretion of IFN-γ, a cytokine that is critically important in controlling bacterial infections (35–37). Since we found that γδ T cells could mediate antibacterial effects in as few as 17 hours after infection (Figure 2c), we examined the role of Vγ2Vδ2 T cells in the IFN-γ response to bacteria. PBMCs were either mock-depleted or depleted of Vδ2 T cells, primed with one of the Vγ2Vδ2 T cell antigens (IBA or pamidronate), and exposed to either heat-killed E. coli or heat-killed M. morganii. After 17 hours, the IFN-γ levels in the supernatants were determined by ELISA. In response to bacteria, PBMCs depleted of Vδ2 T cells produced titers of IFN-γ that were threefold lower than those of mock-depleted PBMCs, showing that optimum production of IFN-γ is critically dependent on Vδ2 T cells (Figure 3a).

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.

These in vitro observations were confirmed in vivo, since SCID mice reconstituted with Vγ2Vδ2 T cell–specific antigen-pretreated PBMC had 3-fold higher levels of serum IFN-γ 17 hours postinfection with E. coli, as compared to SCID mice reconstituted with Vγ2Vδ2 T cell–specific antigen-pretreated PBMC that were first depleted of Vδ2 T cells (Figure 3b). E. coli infection of SCID mice reconstituted with the pretreated PBMCs induced 9.7% of γδ T cells recovered by peritoneal lavage to express intracellular IFN-γ, whereas only 1.6% of γδ T cells from the peritoneal lavage of mock-infected, reconstituted SCID mice expressed intracellular IFN-γ (Figure 3c). It is important to emphasize that neither pamidronate nor IBA had any effect on antibacterial activity or IFN-γ production in the absence of Vδ2 T cells, showing that in this experimental system, the actions of these antigens are Vδ2 T cell–dependent. The data suggest that live bacterial infection in vivo results in IFN-γ production by human Vγ2Vδ2 T cells. Taken together, these data also suggest that IFN-γ production that is at least partially dependent on Vδ2 T cells may be important in mediating antibacterial activity during the first day of bacterial infection in vivo.

Crucial role of Vδ2 T cells and IFN-γ in monocyte-mediated killing of bacteria. One of the dominant mechanisms for eliminating extracellular bacteria is dependent on monocyte-mediated killing, and IFN-γ is an important activator of monocytes (35–37). Therefore, we tested the ability of human γδ T cells to influence monocyte-mediated killing of M. morganii in vitro. Mock-depleted PBMCs or those depleted of Vδ2 T cells were cultured for 4 days in medium containing the live bacterial product IBA. Monocytes from these cultures were then analyzed for their ability to kill M. morganii. Monocytes from PBMC cultures that were mock-depleted of Vδ2 T cells killed more than twice as many bacteria as did monocytes from cultures depleted of Vδ2 T cells. Inclusion of neutralizing mAbs to IFN-γ during the 4-day culture abrogated the Vδ2 T cell–dependent monocyte-mediated killing of M. morganii (Figure 3d), while the addition of IFN-γ to the cultures that were depleted of Vδ2 T cells completely reconstituted monocyte-mediated killing (Figure 3d). Similar results were obtained with E. coli (data not shown). These data suggest that IFN-γ is sufficient to reconstitute γδ T cell–dependent antibacterial activity.

The discovery that marked expansion of γδ T cells occurs in the peripheral blood of patients with bacterial infection first raised the possibility that these T cells play a role in resistance to bacteria (11, 14). This in vivo expansion was repeated in vitro by mixing human PBMCs with bacterial extracts containing nonpeptide antigens that were found to be responsible for expansion of Vγ2Vδ2 T cells in a TCR-dependent manner (16–18, 38, 39). Furthermore, these expanded γδ T cells kill target cells infected with bacteria (40, 41). Abundant evidence from mouse models also suggests that γδ T cells play a very important role in bacterial infection. However, the fact that mouse γδ T cells do not recognize nonpeptide alkylamine or organophosphate antigens secreted by live bacteria raises questions as to whether human γδ T cells play a similar role. Furthermore, it is unknown whether these nonpeptide antigens regulate Vγ2Vδ2 T cell functions in vivo. To address these questions, we developed a chimeric hu-SCID model for studying the effects of human γδ T cells against bacterial infection.

The hu-SCID model has been used widely, and has proven to be a powerful model for study of human cells and tissues (42–46). The fact that SCID mice receiving E. coli infection alone were all dead within 2 days, whereas inoculation with human PBMCs rescued all SCID mice from lethal infection (Figure 1a), demonstrates that inoculated human PBMCs give rise to antibacterial effects. Therefore, the data indicating that hu-SCID mice reconstituted with human PBMCs depleted of Vδ2 T cells had a lower survival rate (Figure 1b) and higher bacterial load (Figure 1c) most likely reflect the function of human Vγ2Vδ2 T cells in vivo. Depletion of Vδ2 T cells markedly decreased, but did not completely abrogate PBMC-mediated resistance to bacterial infection in vivo or monocyte-mediated bactericidal effects in vitro (Figures 1, 2 a–c, and 3d), suggesting that other immune cells contribute to antibacterial effects. In contrast to LPS and dead bacteria, the Vγ2Vδ2 T cell–specific antigen IBA did not activate monocytes, αβ T cells, NK cells, or NK T cells (data not shown), suggesting that distinct antigens orchestrate the responses of different immunologically active cells in host resistance to bacterial infection.

There was no significant expansion of γδ T cells in vivo without antigen pretreatment. Nonetheless, untreated PBMCs that were mock-depleted of Vδ2 T cells provided more protection against infection than did PBMCs that were depleted of Vδ2 T cells (Figure 1b). In general, we obtained better γδ T cell–dependent protection when the PBMCs were pretreated with specific antigen, and therefore we performed most experiments using antigen pretreatment. Consistent with the more potent in vivo protection using primed PBMCs, our unpublished data suggest that γδ T cells in humans may require exposure to dietary and ambient γδ T cell antigens in vivo to enable optimum reactivity in response to bacterial stimulation (Kamath et al., our unpublished observations).

Consistent with the data in clinically infected humans (10–15), our SCID mouse model showed that Vγ2Vδ2 T cells started expanding from day 6 or 7 after bacterial infection to comprise 72% of CD3⁺ T cells in the peritoneal lavage (Figure 2d). Since it is possible that a high percentage of Vγ2Vδ2 T cells might result from a compositional change instead of a cellular expansion, we calculated the absolute numbers of Vγ2Vδ2 T cells harvested from the peritoneal lavage. There were up to 2.5-fold more Vγ2Vδ2 T cells in the peritoneal lavage of hu-SCID mice infected with bacteria than in the peritoneal lavage in uninfected hu-SCID mice (Figure 2e). Whereas the absolute numbers of Vγ2Vδ2 T cells in peritoneal lavage represented only a proportion of these cells, they were detectable in hu-SCID mouse liver, spleen, blood, bone marrow, and other organs of infected mice, suggesting the possibility that these γδ T cells could have local effects, perhaps anti-inflammatory in nature, in these organs at 7 days after infection.

Whereas this in vivo expansion of human γδ T cells in reconstituted, infected SCID mice did not occur until 7 days after infection (Figure 2, d and e), the antibacterial effect of human γδ T cells in our studies was evident as soon as 17 hours after infection (Figure 2c), indicating that expansion of γδ T cells is not required for an antibacterial response. These results are rather surprising, since it is logical to speculate that expansion of γδ T cell numbers occurs for the purpose of subsequently eliminating bacteria. However, a previous report using a mouse model showed that γδ T cell–mediated antibacterial effects against E. coli occur before γδ T cell expansion (47).

Although only 3–15% of Vγ2Vδ2 T cells produced cytokines in response to stimulation with IBA (our unpublished observations), these γδ T cells are much more efficient at cytokine production on a per-cell basis than are αβ T cells (48). Furthermore, these Vγ2Vδ2 T cells, which are reactive to nonpeptide antigens, are 5- to 50-fold more numerous than αβ T cells that are specific for any one peptide, and are thus likely to have a major antibacterial impact. In vitro, Vγ2Vδ2 T cells directly produced cytokines, and also were necessary for optimum IFN-γ production by PBMCs, which was critical for monocyte-mediated killing of bacteria (Figure 3, a, c, and d). In vivo, SCID mice reconstituted with human PBMCs depleted of Vδ2 T cells had lower serum levels of human IFN-γ (Figure 3b), higher mortality (Figure 1b), and higher bacterial loads (Figure 1c) than those receiving PBMCs mock-depleted of Vδ2 T cells.

Besides depletion of fresh Vγ2Vδ2 T cells from PBMCs, other approaches to demonstrate Vγ2Vδ2 T cell functions in vivo are to use either purified fresh human Vγ2Vδ2 T cells or Vγ2Vδ2 T cell lines and clones. However, the requirement for large amounts of human PBMCs for reconstitution, technical difficulties in purifying a small proportion of γδ T cells from whole PBMCs by negative selection, and the unclear fate of γδ T cells after positive selection make the use of purified Vγ2Vδ2 T cells less practical. Vγ2Vδ2 T cell lines and clones, unlike fresh human Vγ2Vδ2 T cells, might have different functions. In a mouse model, expanded mouse γδ T cells kill monocytes during the late stages of infection (49, 50). In contrast, we found in our human studies that γδ T cells, before they are expanded, are necessary for optimum activation of monocytes, illustrating the potential functional differences of expanded and recently stimulated γδ T cells.

Consistent with mouse work, we found that in vitro, certain human Vγ2Vδ2 T cell lines and clones showed different cytokine profiles from fresh Vγ2Vδ2 T cells. These distinctions between fresh and expanded γδ T cells may explain why transfer into SCID mice of expanded Vγ2Vδ2 T cell lines failed to mediate antibacterial activity (our unpublished observation). Perhaps these expanded γδ T cells have a regulatory or anti-inflammatory function later in infection that is distinct from antibacterial activity (50, 51), or they are relatively refractory to antigenic stimulation (51) and lack CD28 (52). Alternatively, expanded γδ T cells may be important in resistance to intracellular bacterial infections, which tend to last longer than extracellular infections.

Vγ2Vδ2 T cells may be considered part of the adaptive immune system, in that they have a memory phenotype, junctionally diverse TCRs that require gene rearrangement for their cell surface expression, and the ability to become either anergic or expanded, depending on the availability of costimulation (18, 53). On the other hand, Vγ2Vδ2 T cells also may be considered part of the innate immune response, since their frequently paired TCR variable region genes Vγ2 and Vδ2 reflect limited germline gene diversity. This V gene pairing enables each Vγ2Vδ2 TCR to immediately recognize families of unprocessed antigens with conserved molecular patterns, such as the alkylamines and prenyl pyrophosphates. This pattern recognition by the Vγ2Vδ2 TCR allows cytokine secretion by large numbers of memory γδ T cells in response to a diverse array of bacterial species. These large numbers of memory T cells that are capable of responding within 2 hours to alkylamine and organophosphate antigens produced by microbes thus may bridge the gap between innate and adaptive immune responses.

Bacterial resistance to antibiotics used prophylactically and empirically is a serious problem that requires new approaches. Our data show that the synthetic aminobisphosphonate, pamidronate, a drug with a proven track record of safety (32, 33), provides prophylaxis and treatment against extracellular bacterial infection in a Vγ2Vδ2 T cell–dependent manner. Based on our findings, synthetic Vγ2Vδ2 T cell–specific antigens could potentially be used in humans to treat or prevent bacterial infection, as an alternative to antibiotics.

In summary, these data suggest that Vγ2Vδ2 T cells are likely to play an important role in mediating natural resistance to gram-positive and gram-negative extracellular bacterial infections in humans. Administration of antigens specifically recognized by this population of human γδ T cells may be useful in the prevention and treatment of bacterial infection in humans.

We thank Craig Morita for his critical reading of the manuscript. This research was supported by grants from NIH and the Arthritis Foundation.

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