γδ T lymphocytes have been shown to regulate immune responses in diverse experimental systems. Because distinct γδ T cell subsets, as defined by the usage of certain TCR V genes, preferentially respond in various diseases and disease models, we have hypothesized that the various γδ T cell subsets carry out different functions. To test this, we compared one particular γδ T cell subset, the Vγ1+ subset, which represents a major γδ T cell type in the lymphoid organs and blood of mice, to other subsets and to γδ T cells as a whole. Using Listeria monocytogenes infection as an infectious disease model, we found that bacterial containment improves in mice depleted of Vγ1+ γδ T cells, albeit mice lacking all γδ T cells are instead impaired in their ability to control Listeria expansion. Our findings indicate that Vγ1+ γδ T cells reduce the ability of the innate immune system to destroy Listeria, even though other γδ T cells as a whole promote clearance of this pathogen.

Although the immunological role of the γδ T lymphocytes is not well understood, several studies have now shown that they influence the development of inflammatory lesions in certain diseases. However, in some experimental disease models in mice, γδ T cells appear to reduce inflammatory damage, whereas in others, results instead imply that they exacerbate it. For example, whether γδ T cells were experimentally ablated by genetic manipulation or depletion with a specific mAb, increased or accelerated inflammatory damage of host tissue was seen in the liver of mice infected with Listeria monocytogenes (1, 2), in the lungs of mice infected with Mycobacterium tuberculosis (3), and in the testes of mice with induced autoimmune orchitis (4), implying that γδ T cells normally limit inflammation. In contrast, in coxsackievirus B3-induced myocarditis, the γδ T cells appeared to enhance the inflammation, because transfer of the γδ T cells infiltrating the heart of a susceptible infected mouse resulted in heart inflammation in a normally myocarditis-resistant strain following infection (5). In a mouse candidiasis model, γδ T cells also appeared to have proinflammatory effects, inducing NO production by macrophages and contributing to clearance of the pathogen (6). Conversely, two additional studies attributed both pro- and anti-inflammatory effects to γδ T cells within a single disease model, dependent upon the time point at which the γδ T cells were examined. Specifically, in mice with collagen-induced arthritis, removal of γδ T cells at early stages of the disease reduced the incidence and severity of the ensuing joint inflammation, whereas removal of γδ T cells later caused a rapid onset of severe arthritis (7). A time-dependent dual effect of γδ T cells was also reported in a mouse model of spontaneous abortion (in DBA-2-mated CBA/J mice), in which γδ T cells were found to have a proinflammatory role early in the pregnancy, infiltrating the uterus and producing Th1 cytokines that promote spontaneous abortion, but an anti-inflammatory effect later, switching to Th2-type cytokine production and protecting against abortion (8). Consistently, γδ T cells have been shown to produce Th1 (proinflammatory) cytokines during infection with one type of pathogen, the intracellular bacterium L. monocytogenes, but to produce Th2 (anti-inflammatory) cytokines in mice with another, Nippostrongylus brasiliensis (9). Together, these observations show that one cannot simply regard γδ T cells as either pro- or anti-inflammatory; instead, they appear to be capable of playing either role, depending upon unknown influences.

The preferential tissue localization of some γδ T cells expressing particular Vγ and/or Vδ genes and the finding that certain Vγ-Vδ pair combinations frequently recur among nonclonal γδ T cells (reviewed in Ref. 10) have led to a tendency to regard γδ T cells as subsets based on their expression of certain Vγ and/or Vδ genes. Although a number of γδ T cell clones have been identified that respond to certain specific Ags (11, 12, 13, 14, 15), in many cases, the type of Vγ gene expressed corresponds to the cell’s ability to respond to certain Ags, even though other components of the TCR may differ. For example, in the mouse, members of the Vγ5/Vδ1 subset, which resides primarily in the skin, typically respond to what appears to be an autoantigen expressed by keratinocytes (16). In addition, we and others have found preferential responses of the mouse Vγ6/Vδ1+ subset at various anatomical locations during inflammation induced in a variety of ways: by L. monocytogenes infection (17, 18), autoimmune orchitis (18, 19), experimental allergic encephalomyelitis (20), and Escherichia coli infection (21). Thus, the Vγ6/Vδ1+ subset probably responds to an inflammation-induced host Ag as well. Such responses by these two subsets may be predictable because they each express invariant canonical TCRs. However, polyclonal responses have also been seen among γδ T cell subsets with more diverse TCRs, such as the junctionally variable Vγ9/Vδ2+ blood human γδ T cells, which strongly proliferate in response to mycobacteria during in vitro culture (reviewed in Ref. 22). In addition, previous studies of our own and of others involving the mouse Vγ1 subset (23, 24, 25, 26, 27), which expresses a junctionally diverse Vγ1-Jγ4-Cγ4 γ chain generally together with either Vδ6 or Vδ4, revealed an apparent autoreactivity. Specifically, Vγ1+ γδ T cells spontaneously release cytokine at low to moderate levels when simply grown by themselves in culture medium, in the absence of any deliberately added Ag or accessory cells. Moreover, transfection of a TCR-negative T cell line with a construct containing genes encoding a Vγ1-Jγ4-Cγ4-containing TCR gave rise to new cell line that spontaneously released cytokine, indicating that the Vγ1 TCR confers this response (28). Recently, we have found that reducing or eliminating serum in the medium greatly increases this response, possibly indicating that the hybridomas respond to a stress-induced self protein (C. T. Cady and W. K. Born, unpublished observations), which may be related to the mycobacterial stress-inducible protein heat shock protein-60, that augments the responses of this subset (12, 23).

The responses of different γδ T cell subsets have been shown to occur at specific time points during infection (29, 30) or to vary depending upon the severity of inflammation induced (31). These findings taken together with the ability of γδ T cells to respond to Ags as entire subsets led us to propose that the function of a γδ T cell may be dictated by the TCR it bears. Studies previously demonstrating that γδ T cells can reduce or prevent host inflammatory damage (1, 2, 4, 7) moreover predict that certain γδ T cell subsets might actually hinder the host response to an infectious agent. In this study we present evidence showing that whereas clearance of L. monocytogenes in infected mice is negatively affected if all types of γδ T cells are depleted, removing only the Vγ1+ subset is, in contrast, beneficial in this disease model. (Note that the nomenclature for the murine Vγ genes in this paper follows that set forth by S. Tonegawa’s laboratory (32).)

Both male and female mice were used in these experiments at 8–12 wk of age. For any given experiment, age- and sex-matched mice were used, with three to five mice per group except as noted in Fig. 4 and Table I. C57BL/10J, CB.17/SCID, and C57BL/6-TCRβ gene-targeted mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and either used directly or bred in our facility. C57BL/6 mice and C3H/HeN mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and bred in our facility. The TCR-Vγ4/6 gene-targeted mice (33) were back-crossed three times with C57BL/10 mice, then interbred, and offspring homozygous for the Vγ4/6 null allele were identified by Southern blotting. Vγ4/6−/− mice on the C3H/HeN background were similarly generated after five backcrosses. Mice homozygous for the Vγ4/6 null allele were then interbred to generate the Vγ4/6−/− mice used in this study. Mice lacking functional TCR-Cδ genes and having the C57BL/10 background were also generated at the same time from C57BL/6-TCRδ−/− progenitors (bred in our facility from Jackson Laboratory stock), as controls, after four backcrosses onto the C57BL/10 background.

FIGURE 4.

Levels of L. monocytogenes in the spleens of mAb-treated TCRβ−/− mice vs sham-treated controls. C57BL/6-TCRβ−/− mice were infected and treated as described in Fig. 1; a, b, and c represent independent experiments. (Note that error bars in c show SDs, but in the anti-Vγ1-treated group in b instead show the actual range of the data, because only two animals in these groups were available for analysis at the time of the experiment.) The Listeria doses were: a, 2.4 × 104; b, 2.4 × 104; and c, 2.0 × 104.

FIGURE 4.

Levels of L. monocytogenes in the spleens of mAb-treated TCRβ−/− mice vs sham-treated controls. C57BL/6-TCRβ−/− mice were infected and treated as described in Fig. 1; a, b, and c represent independent experiments. (Note that error bars in c show SDs, but in the anti-Vγ1-treated group in b instead show the actual range of the data, because only two animals in these groups were available for analysis at the time of the experiment.) The Listeria doses were: a, 2.4 × 104; b, 2.4 × 104; and c, 2.0 × 104.

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Table I.

Distribution of γδ T cell subsets in normal vs Vγ4/6−/− mice

StrainUninfectedInfected
% Vγ1+% Vγ4+% Vγ othera% Vγ1+% Vγ4+% Vγ other
C3H/HeN 70.8 (±1.6) 17.1 (±3.6) [<12.1] 79.6 (±2.9) 8.4 (±1.3) [<12] 
C3H/HeN Vγ4/6−/− 79.3 (±1.5) – 20.7 (±1.5) 84.6 (±1.3) – 15.2 (±1.3) 
C57BL/10 70.0 (±7.8) 20.9 (±3.4) [<9.1] 62.1 (±7.1) 27.6 (±4.8) [<10.3] 
C57BL/10 Vγ4/6−/− 80.6b – 19.4b 64.6 (±4.1) – 35.5 (±4.1) 
StrainUninfectedInfected
% Vγ1+% Vγ4+% Vγ othera% Vγ1+% Vγ4+% Vγ other
C3H/HeN 70.8 (±1.6) 17.1 (±3.6) [<12.1] 79.6 (±2.9) 8.4 (±1.3) [<12] 
C3H/HeN Vγ4/6−/− 79.3 (±1.5) – 20.7 (±1.5) 84.6 (±1.3) – 15.2 (±1.3) 
C57BL/10 70.0 (±7.8) 20.9 (±3.4) [<9.1] 62.1 (±7.1) 27.6 (±4.8) [<10.3] 
C57BL/10 Vγ4/6−/− 80.6b – 19.4b 64.6 (±4.1) – 35.5 (±4.1) 
a

The percentage of γδ T cells expressing Vγs other than Vγ1, Vγ4, or Vγ6 was calculated by subtraction. For mice carrying normal Vγ4 and Vγ6 genes, the average of the non-Vγ1/Vγ4+ population is shown in brackets for comparison; the actual Vγ1/4/6-negative population is less than the value shown in brackets because it includes Vγ6+ cells.

b

, Average obtained with samples from two mice, analyzed independently. All others shown are averages obtained from three to six mice, ±SD (in parentheses).

L. monocytogenes, strain EGD, was freshly grown from frozen stocks on a shaker in tryptose phosphate broth overnight at 37°C and used the same day. Mice were infected by an i.v. injection of ∼1/10th the 50% lethal dose of L. monocytogenes by inoculating 0.2 cc of bacteria diluted in sterile HBSS into the tail vein (for exact doses in individual experiments, see figure legends). An i.v. rather than an i.p. route of infection was chosen for these experiments because it gave less variability in the number of bacteria in individual infected mice than did i.p. infection. Mice were sacrificed on day 3 of the infection, and the spleen and in some experiments a section of the main liver lobe were removed from each. Livers were directly homogenized in sterile water with a motor-driven pestle, whereas spleens were first dispersed in HBSS on stainless steel screens and divided into two equal portions, one for determining bacterial content and the other for flow cytometry (see below). The portion reserved for determining bacterial levels was then further homogenized with a motorized pestle. Spleen and liver homogenates were diluted sequentially in sterile water, and 100 μl of selected dilutions were plated on trypticase soy agar plates. Following overnight incubation at 37°C, colonies were counted, and the number of bacteria per spleen, or for liver per gram of wet tissue, was calculated. In some experiments a single animal contained Listeria levels that were not representative of the others within the same experimental group (2 or more SD above or below the mean). Such outliers are probably caused by unknown health differences in the starting population, and these were eliminated from the analysis. All experiments were conducted at least twice; figures show results from a representative experiment. In some experiments, hematoxylin/eosin-stained sections of liver were also examined.

Three to 5 days before infection, mice to be depleted of certain γδ T cells by mAb treatment were injected with 250 μg of the appropriate purified mAb in a volume of 0.2 cc in sterile HBSS via the tail vein. Three different Abs were used: anti-Vγ1 (clone 2.11) (34), anti-Vγ4 (clone UC3) (35), and anti-Cδ (clone GL3) (36). Note that mAb 2.11 does not stain hybridomas expressing a TCR containing the closely related Vγ2 chain (data not shown) and therefore appears to be specific for Vγ1. The mAbs were produced either by ascites tumor growth in Pristane-primed C.B-17/SCID mice (anti-Vγ4 and anti-Cδ) or by in vitro cell culture (anti-Vγ1) and were purified by column chromatography using either protein A (anti-Cδ) or protein G (anti-Vγ1 and anti-Vγ4)-Sepharose (Pharmacia, Piscataway, NJ). All these mAbs were originally generated in the hamster and are IgG mAbs. Thus, undepleted control mice were similarly sham-treated with 250 μg of purified normal hamster serum IgG (Ham IgG;3 Jackson ImmunoResearch Laboratories, West Grove, PA). To remove potential Ab aggregates in the preparations, just before injection the Abs were incubated for 10 min at 37°C, then spun at 14,000 rpm in a microfuge for 10 min at room temperature, and all but the bottom ∼50 μl were transferred to a fresh sterile tube. Depletions of the relevant cells are usually apparent using these mAbs within 24 h and remain effective for at least 2 wk (data not shown; longer time periods were not tested).

Anti-CD3 mAb KT3 (37) as well as anti-Vγ1, -Vγ4, and -Cδ mAbs were purified as described above and conjugated to biotin or, for anti-Cδ, to fluorescein. PE-streptavidin (Tago Immunologics BioSource, Camarillo, CA) was used as a secondary reagent to detect binding of the biotin-labeled mAbs. Precalibrated amounts of each preparation were used to examine splenic T cell populations in mice previously injected with mAbs to deplete certain subsets, using two-color flow cytometry. The staining was conducted essentially as previously described (38); in brief, cell suspensions from half of each spleen were treated with Gey’s solution to lyse the RBC, then passed over nylon wool columns to enrich for T cells. For each staining, between 105 and 106 cells/well in a 96-well plate were first incubated with unlabeled mAb 2.4G2 (39) to reduce nonspecific mAb binding via Fc receptor, then washed, incubated with a biotinylated mAb, washed again, and incubated with an FITC-labeled mAb together with PE-streptavidin. After final washing, the cells were analyzed on a Coulter XL (Coulter, Miami, FL) to confirm the efficacy of the depletion. Spleen cells from at least one mouse in each experimental group were examined to verify depletion in every experiment. This was necessary because the depletion requires intact Ab (40), and the inadvertent use of a partially degraded or denatured Ab stock could result in false negatives. Fig. 1 shows typical results from mice treated in vivo with anti-Vγ1, -Vγ4, and -Cδ together with results from sham-treated controls. As shown, although the depletion is probably never 100% complete, the few positive cells that escape depletion generally show quite low levels of TCR and are thus presumably functionally impaired. The absence of Vγ4+ cells in the Vγ4/6−/− mice is also documented in Fig. 1 D. A lack of Vγ6+ cells in the same mice cannot be similarly verified because an anti-Vγ6 mAb is not yet available.

FIGURE 1.

FACS profiles of mAb-treated, L. monocytogenes-treated mice. C57BL/10 mice were depleted of the desired γδ T cell type by injecting them i.v. with 250 μg of the appropriate mAb. Three to 5 days later mice were infected by i.v. injection of ∼4 × 104L. monocytogenes. On day 3 of the infection spleens were harvested, the T cells were enriched by nylon wool passage, and the expressed TCRs were determined by FACS. A, Splenic T cells from mice sham-treated with Ham IgG (top) or treated with anti TCR-Cδ mAb (bottom; 10,000 cells total). B, Splenic T cells from mice sham-treated with Ham IgG (top) or treated with anti-TCR-Vγ4 mAb (bottom; 25,000 cells total). C, Splenic T cells from mice sham-treated with Ham IgG (top) or treated with anti-TCR-Vγ1 mAb (bottom; 25,000 cells total). D, Splenic T cells from a normal C57BL/10 mouse (top) or a C57BL/10 Vγ4/6−/− mouse (bottom; 25,000 cells total).

FIGURE 1.

FACS profiles of mAb-treated, L. monocytogenes-treated mice. C57BL/10 mice were depleted of the desired γδ T cell type by injecting them i.v. with 250 μg of the appropriate mAb. Three to 5 days later mice were infected by i.v. injection of ∼4 × 104L. monocytogenes. On day 3 of the infection spleens were harvested, the T cells were enriched by nylon wool passage, and the expressed TCRs were determined by FACS. A, Splenic T cells from mice sham-treated with Ham IgG (top) or treated with anti TCR-Cδ mAb (bottom; 10,000 cells total). B, Splenic T cells from mice sham-treated with Ham IgG (top) or treated with anti-TCR-Vγ4 mAb (bottom; 25,000 cells total). C, Splenic T cells from mice sham-treated with Ham IgG (top) or treated with anti-TCR-Vγ1 mAb (bottom; 25,000 cells total). D, Splenic T cells from a normal C57BL/10 mouse (top) or a C57BL/10 Vγ4/6−/− mouse (bottom; 25,000 cells total).

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C57BL/10 mice depleted of TCR-γδ+ cells by prior i.v. injection of anti-Cδ mAb showed some impairment in their ability to clear Listeria on day 3 of the infection as previously reported (2, 41). Consistently in these experiments, the degree of impairment was smaller than that in a previous study (2), possibly due to the choice of an i.v. rather than an i.p. route of infection. However, in contrast, mice similarly depleted of Vγ1+ γδ T cells only showed an improvement in bacterial clearance (see Fig. 2). The improvement ranged from a 3-fold to ∼16-fold reduction in various experiments. In contrast, depletion of Vγ4+ γδ T cells had no apparent effect.

FIGURE 2.

Levels of bacteria in the spleens of mAb-treated C57BL/10 L. monocytogenes-infected mice. Mice were treated and infected with 5.4 × 104L. monocytogenes as described in Fig. 1.

FIGURE 2.

Levels of bacteria in the spleens of mAb-treated C57BL/10 L. monocytogenes-infected mice. Mice were treated and infected with 5.4 × 104L. monocytogenes as described in Fig. 1.

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Fig. 3 shows the results of similar experiments conducted with several different mouse strains. Both C57BL/10 and C57BL/6 mice were examined because, despite their many similarities, these two strains carry different TCR-γ gene alleles, which render slight differences in the Vγ1 chains they express (25). As shown, however, depletion of Vγ1+ γδ T cells was beneficial for clearance of Listeria in both of these strains. (Note that in Fig. 3,b, the difference between the Ham IgG sham-treated group and the anti-Vγ1-treated group is significant, p ≤ 0.10.) Both the C57BL/10 and C57BL/6 strains are relatively resistant to infection by Listeria; we therefore examined in addition two Listeria-sensitive strains to test whether Vγ1+ γδ T cells play a similar role. As shown in Fig. 3, for C3H/HeN mice the beneficial effect of Vγ1+ cell removal was still evident, although for BALB/c mice we could not demonstrate it. The reason for Listeria sensitivity in the C3H/HeN strain is not known, but in BALB/c mice it may be due to the inherent bias of these mice to overproduce IL-4 and generate Th2-type immune responses (42). Because C3H/HeN and BALB/c are different in this regard, it is perhaps not surprising that they also respond differentially to the removal of Vγ1+ cells.

FIGURE 3.

Levels of bacteria in the spleens of mAb-treated L. monocytogenes-infected mice of Listeria-resistant and -sensitive strains. C57BL/10 and C57BL/6 are Listeria-resistant strains, whereas C3H/HeN and BALB/c are Listeria-sensitive strains. Mice were treated and infected as described in Fig. 1. Listeria doses were as follows: a, 4 × 104; b, 5.4 × 104; c, 4 × 102; and d, 5.2 × 102. (Note that in b, the difference between the Ham IgG sham-treated group and the anti-Vγ1-treated group is significant; p ≤ 0.10, using Student’s t test.)

FIGURE 3.

Levels of bacteria in the spleens of mAb-treated L. monocytogenes-infected mice of Listeria-resistant and -sensitive strains. C57BL/10 and C57BL/6 are Listeria-resistant strains, whereas C3H/HeN and BALB/c are Listeria-sensitive strains. Mice were treated and infected as described in Fig. 1. Listeria doses were as follows: a, 4 × 104; b, 5.4 × 104; c, 4 × 102; and d, 5.2 × 102. (Note that in b, the difference between the Ham IgG sham-treated group and the anti-Vγ1-treated group is significant; p ≤ 0.10, using Student’s t test.)

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Mice infected with Listeria mount an αβ T cell-mediated adaptive immune response that is only first weakly detectable on the third day of infection (43). The detrimental effect of removing Vγ1+ cells during a Listeria infection at an early time point thus suggested that they primarily influence components of the innate immune response. Thus, we tested whether mice incapable of producing Listeria-immune αβ T cells (TCRβ−/− mice) also showed an improvement in bacterial clearance in the absence of this subset. As shown in Fig. 4, a decrease in susceptibility to Listeria was likewise evident in mice lacking αβ T cells.

We had observed that removing Vγ4+ γδ T cells had, in contrast to removing Vγ1+ cells, no effect on listerial clearance (see Figs. 2 and 3,a). We next depleted C57BL/6 mice simultaneously of both Vγ1 and Vγ4+ subsets (thus eliminating or inactivating ∼85% of the γδ T cells present in blood and spleen) and tested the effect of this on Listeria clearance. As shown in Fig. 5, the benefit of removing Vγ1+ cells largely disappeared when Vγ4+ cells were also removed. Thus, the Vγ4+ subset appears to play a role as well, one that seems to have an outcome opposite that of the Vγ1+ subset. However, the effect of eliminating both these subsets is to return bacterial clearance to approximately the wild-type level. We had expected that removal of both subsets would instead exacerbate the infection, because removing all γδ T cells has this effect, and very few γδ T cells remain after depleting both these subsets. This finding implies that removing another, less abundant γδ T cell subset must also occur to produce a detrimental effect. We have previously noted a preferential increase in the percentage of Vγ6+ γδ T cells in the liver of mice with experimental listeriosis (17) and therefore suspected that the Vγ6+ γδ T cell subset might be the one in question.

FIGURE 5.

Levels of L. monocytogenes in the spleens of mice simultaneously depleted of both Vγ1+ and Vγ4+ γδ T cells. C57BL/6 mice were infected and treated as described in Fig. 1; those treated with both anti-Vγ1 and -Vγ4 received a total of about 500 μg of total Ig each; those treated with only one mAb received ∼250 μg of total Ig. The Listeria dose was 5.4 × 104. (Note that the difference observed between the anti-Vγ1-treated and anti-Vγ1/anti-Vγ4-treated groups is significant, p ≤ 0.03, using Student’s t test.)

FIGURE 5.

Levels of L. monocytogenes in the spleens of mice simultaneously depleted of both Vγ1+ and Vγ4+ γδ T cells. C57BL/6 mice were infected and treated as described in Fig. 1; those treated with both anti-Vγ1 and -Vγ4 received a total of about 500 μg of total Ig each; those treated with only one mAb received ∼250 μg of total Ig. The Listeria dose was 5.4 × 104. (Note that the difference observed between the anti-Vγ1-treated and anti-Vγ1/anti-Vγ4-treated groups is significant, p ≤ 0.03, using Student’s t test.)

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Because an anti-Vγ6 mAb is not yet available, we could not simply deplete mice of Vγ6+ cells to examine the role of this subset. However, mice incapable of producing either Vγ4 or Vγ6+ γδ T cells were previously generated by gene targeting (33). We therefore backcrossed these mice onto the C57BL/10 and C3H/HeN backgrounds to generate lines with nearly homogenous backgrounds and tested them for their ability to clear Listeria. C57BL/10 background, TCRδ−/− mice, which contain no γδ T cells, were similarly infected at the same time as controls. As shown in Fig. 6, for mice with the C3H/HeN background, removing both the Vγ4 and Vγ6+ subsets had a small, but clearly detrimental, effect on Listeria containment in spleen and liver (the Vγ4/6−/− mice, on the average, contained ∼3 times more bacteria in spleen and ∼7 times more in liver than did wild-type mice; Fig. 6 a). However, on the C57BL/10 background, Vγ4/6−/− mice were indistinguishable from wild-type mice in the numbers of bacteria remaining in spleen or liver.

FIGURE 6.

Levels of L. monocytogenes in the spleens and livers of mice lacking Vγ4+ and Vγ6+ cells compared with normal controls. Mice are described in Materials and Methods and were infected as described in Fig. 1. a, The Listeria dose was 4.5 × 102. b, The Listeria dose was 4.1 × 104.

FIGURE 6.

Levels of L. monocytogenes in the spleens and livers of mice lacking Vγ4+ and Vγ6+ cells compared with normal controls. Mice are described in Materials and Methods and were infected as described in Fig. 1. a, The Listeria dose was 4.5 × 102. b, The Listeria dose was 4.1 × 104.

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We went on to examine the distribution of γδ T cell subsets in spleens of infected Vγ4/6−/− vs wild-type mice (see Table I) and found that cells from mice of the C3H/HeN background were quite differently affected than those of the C57BL/10 background. Specifically, the percentage of Vγ1+ cells increased in C3H/HeN, but decreased in C57BL/10, a pattern that was also seen in both strains in the absence of the Vγ4 and Vγ6 subsets. The Vγ4+ subset showed exactly the opposite pattern, decreasing in percentage in C3H/HeN, but increasing in C57BL/10. Cells expressing Vγ genes other than 1, 4, and 6 (Vγ other in Table I) in the Vγ4/6−/− mice behaved similarly to the Vγ4+ subset in wild-type mice, decreasing in relative abundance on the C3H/HeN background, but increasing on the C57BL/10 background. These changes in percentage of Vγ1 and Vγ4 subsets in the wild-type mice were similar to our results in a previous study comparing the same two strains (44), and the increase in the percentage of Vγ1+ cells in C3H/HeN might by itself explain why this strain is adversely affected by the lack of Vγ4+ and Vγ6+ cells, whereas C57BL/10 is seemingly not.

However, when we looked at the effect of infection on the actual numbers of Vγ1 and Vγ4 subsets, an additional difference between the strains was revealed; whereas the Vγ1+ T cells in C3H/HeN mice increased in number, those in C57BL/10 mice decreased markedly (∼10-fold; see Table II). Induction of apoptosis in the lymphocytes of C57BL/6 and BALB/c mice has been previously noted using high Listeria doses early in infection (45), and in our hands, splenic T cell yields, on the average, decrease to about 25% of the normal level in C57BL/6 and C57BL/10 mice, whereas they increase slightly in C3H/HeN under the experimental conditions used in this study (data not shown). Whether C3H/HeN mice given comparable doses of Listeria would also show T cell apoptosis is not known and could not be examined here because this Listeria-sensitive strain would die before day 3 of infection if given doses at the levels used here for C57BL/10 mice. In both strains, however, we found that the Vγ4+ cells behaved differently from the Vγ1+ cells, showing no increase in numbers in C3H/HeN, and in C57BL/10 mice a lesser tendency to disappear than Vγ1+ cells (about equal to that of T cells in general). Surprisingly, the abundance of the Vγ1+ subset after infection depended in both strains on whether they were Vγ4/6−/−, because C3H/HeN Vγ4/6−/− mice showed virtually no increase in Vγ1+ cells, whereas C57BL/10 Vγ4/6−/− mice showed an even greater loss of the Vγ1+ subset than did the wild-type controls. In contrast, cells expressing Vγ genes other than 1, 4, and 6 in Vγ4/6−/− mice increased in C3H/HeN background mice and decreased in Vγ4/6−/− mice of the C57BL/10 background. Thus, deletion of both the Vγ4+ and Vγ6+ subsets appears to affect the two strains differently not only in terms of bacterial clearance, but also in the responses evoked in the other γδ T cell subsets. Whether γδ T cell subset response differences can explain the bacterial clearance difference remains to be determined; certainly, other factors dictated by genetic disparities between the strains may be involved as well.

Table II.

Changes in Vγ frequency in infected mice

StrainInfected/Uninfected
Vγ1 ratioVγ4 ratioVγ other ratio
C3H/HeN 2.32 (1.80–2.82) 1.00 (0.95–1.05) ND 
C3H/HeN Vγ4/6−/− 1.14 (0.78–1.44) – 1.73 (1.14–2.87) 
C57BL/10 0.10 (0.05–0.18) 0.24 (0.07–0.54) ND 
C57BL/10 Vγ4/6−/− 0.05 (0.03–0.08) – 0.12 (0.08–0.15) 
StrainInfected/Uninfected
Vγ1 ratioVγ4 ratioVγ other ratio
C3H/HeN 2.32 (1.80–2.82) 1.00 (0.95–1.05) ND 
C3H/HeN Vγ4/6−/− 1.14 (0.78–1.44) – 1.73 (1.14–2.87) 
C57BL/10 0.10 (0.05–0.18) 0.24 (0.07–0.54) ND 
C57BL/10 Vγ4/6−/− 0.05 (0.03–0.08) – 0.12 (0.08–0.15) 
a

Ratios shown represent, for each subset, the average number of cells obtained per spleen in infected mice divided by that obtained in uninfected mice. The range calculated from the SD obtained for each population is shown beside each ratio.

The experiments in this study indicate that mice infected with L. monocytogenes actually improve in their ability to clear bacteria when they lack Vγ1+ γδ T cells, even though depleting all γδ T cells has a detrimental effect. Although such findings are not unprecedented (46), this is surprising given that removing a particular lymphocyte subset from a mouse might be regarded as impairing its immune system. A trivial explanation for this finding would be that the anti-Vγ1 mAb used to remove the Vγ1+ cells cross-reacts with the Listeria bacteria and causes their elimination. This is unlikely not only because Listeria largely propagate intracellularly and thus escape humoral defense mechanisms, but also because the effect was absent in BALB/c mice (Fig. 3), and joint depletion of the Vγ4+ subset at the same time reversed the beneficial effect of depleting Vγ1+ cells (Fig. 5). At this time we do not understand how the Vγ1 cell-dependent effect is mediated. It may be due to an alteration in Th1 vs Th2 cytokine profiles in the course of the infection, and indeed others have shown that at least some Vγ1+ γδ T cells preferentially produce IL-4 (47) and might in this way alter the type of αβ T cell response that is elicited. Because mice that cannot produce αβ T cells are similarly affected, it seems more likely that the Vγ1+ cells themselves directly influence the ability of macrophages and/or neutrophils to kill Listeria. γδ T cells that are stimulated to secrete IFN-γ have been shown to enhance the ability of macrophages to kill Candida albicans (6), so it may be that IL-4-producing Vγ1+ cells stimulated during a Listeria infection, in contrast, down-regulate macrophage function. Alternatively, or as well, the Vγ1+ cells might indirectly influence macrophage activity by causing other T cells to produce IL-4 through secreting other cytokines. This could be brought about by the chemokine monocyte chemoattractant protein-1, which has been shown to promote IL-4 production in T cells cultured in vitro (48), because γδ T cells were previously found to be necessary for the production of monocyte chemoattractant protein-1 during Listeria infection (49). In a recent study by Egan and Carding, activated macrophages during Listeria infection were found to stimulate mouse γδ T cells, particularly those expressing Vγ1, to acquire cytolytic activity and then to lyse the activated macrophages (50), which suggests yet a third scenario by which Vγ1+ cells might decrease macrophage activity.

In any case, if Vγ1+ cells indeed down-regulate macrophage function, one might predict that in other situations, their depletion would instead be counterproductive and could lead to inflammatory damage. In fact, we have already found that in coxsackievirus B3 infection, depletion of Vγ1+ γδ T cells does exactly this, leading to an exacerbation of the myocarditis evoked by infection with the virus (57). In contrast, removal of the Vγ4 subset reduces inflammatory damage in coxsackievirus B3-induced myocarditis. Our observation that concomitant Vγ4+ cell depletion reverses the beneficial effect of depleting Vγ1+ cells in listeriosis also supports the idea that the two subsets carry out opposing roles. Thus, if Vγ1+ cells decrease macrophage activity, the Vγ4+ cells, in contrast, might increase it, and if these two subsets normally balance one another out, deleting both would be expected to restore the status quo, as we indeed observed. Although we did not see a decrease in bacterial killing in Vγ4 cell-depleted mice, as might be expected if they indeed up-regulate macrophage activity, the enhancing effect of Vγ4+ cells might be substantially less important in bacterial killing than is the down-regulatory effect of the Vγ1+ cells, at least in the Listeria system. Further study will be needed to determine this.

Whether the function of the Vγ6 subset differs from that of the Vγ1 and Vγ4 subsets is still uncertain. Although we found that C3H/HeN Vγ4/6−/− mice showed an impairment in bacterial clearance compared with wild-type controls (Fig. 6,a), this was not true of Vγ4/6−/− mice on the C57BL/10 background. We did note differences between these mice within the remaining γδ T cells during infection (Table I); in particular, although Vγ1+ cells in C3H/HeN Vγ4/6−/− mice were present in comparable numbers in both infected and infected mice, in C57BL/10 Vγ4/6−/− mice the infection resulted in a 20-fold reduction in Vγ1+ cells. Further study will be necessary to determine whether this alone can explain the difference in relative resistance of Vγ4/6−/− mice of the C3H/HeN vs the C57BL/10 background.

In mice lacking all γδ T cells, not only bacterial containment but also an increase in the frequency of necrotic liver lesions have been previously reported by both ourselves (2) and others (1). In the present study alterations in histopathology were rarely seen in animals depleted of all types of γδ T cells via in vivo mAb injection, although these were clear when using TCRδ−/− mice. Perhaps this is due to our inability to completely eliminate each cell type with mAb-mediated depletion or was less obvious due to our choice of an i.v. rather than an i.p. route of infection as used in the previous studies. In support of the former argument, we, in fact, in general find that the effect of removing all γδ T cells by disruption of the Cδ gene has a stronger effect than does depleting γδ T cells with an mAb (for example, see Fig. 6 b, in which TCRδ−/− mice show ∼27 times more bacteria in liver and ∼4 times more in spleen compared with wild-type mice), perhaps because genetic inactivation is 100% effective. We also failed to note any histological differences in the livers of mice depleted of Vγ1+ or Vγ4+ γδ T cells only or among Vγ4/6−/− mice on either the C57BL/10 or C3H/HeN background. Thus, it is possible that this phenomenon depends on the absence of several different γδ T cell subsets.

We have focused in this study solely on Vγ gene expression, ignoring the impact of the coexpressed Vδ. This decision, based on our observations of common response patterns among γδ T cells expressing Vγ1 without apparent limitation from Vδ, could nonetheless be biasing our findings. In particular, Pereira et al. have previously reported a largely Vγ1+ subset that in the DBA/2 strain usually coexpresses a Vδ6.3 allele, is Thy-1low in the thymus, displays restrictions in TCR diversity, and has a tendency to produce IL-4 (45, 47, 51). Therefore, in a preliminary experiment we attempted to deplete only this portion of the Vγ1 subset using a Vδ6.3 mAb (52) (which should be possible, because virtually all Vδ6.3 cells in C57BL/10 mice coexpress a Vγ1 chain (53)); this had a slightly detrimental effect on bacterial clearance, a result opposite to what we expected (data not shown). However, at this time it is unclear whether the properties of the Vδ6.3+ cells described by Pereira et al. are peculiar to DBA/2 mice and may be absent or less pronounced in the C57BL/10 strain.

The technique of cell depletion via mAb injection is often considered to be less stringent than genetic inactivation, particularly when using anti-TCR mAbs, because the treatment also usually transiently activates the cells of interest. Cytokines elicited by anti-TCR mAb treatment generally fall to normal levels in the serum within 48 h after injection, however (54), so to circumvent this problem, we injected mice with mAb 3–5 days before infecting them with Listeria. As well, all the mAbs used, anti-Vγ1, -4, and TCR-δ, are hamster-derived IgG Abs, and thus any Fc receptor-driven side effects they have should be similar. Thus, even if transient activation rather than depletion produced the effects seen in the study described here, our observations indicate that stimulation of different γδ T cell subsets results in the production of different cytokines. We believe that the effect is instead due to cell depletion, because in a related study using coxsackievirus B3 infection in mice (57), we found that reconstituting TCRδ−/− mice with γδ T cell populations first depleted in vitro of certain subsets produced effects in accordance with those obtained using direct in vivo depletion. Because cytokine production would not be triggered using the reconstitution protocol, the simplest explanation is that the depletion is responsible for the observed effect in both cases. We are now in the process of generating Vγ1 and Vγ6 gene-inactivated mice to both verify and expand our analyses.

In a recent report from Nakamura et al. (55) in which γδ T cell function in Listeria-infected C3H/HeN mice was examined, the depletion of Vγ1+ cells was found to be detrimental to bacterial clearance, a result in exact opposition to that presented here. Because the mouse strain was the same as one of the strains used here, differences in protocols used in the two studies (i.e., Nakamura et al. used a much lower infectious dose and a different route of infection and examined bacterial levels at a later time point) could have implications for the actual timing and/or stimulation of γδ T cell regulatory responses.

The findings presented here support the hypothesis that the type of TCR borne by a γδ T cell predisposes it toward a particular function. Because many of the TCRs that arise on γδ T cells are developmentally predetermined at least to some degree, the idea that their function might be “hardwired” along with the TCR is actually consistent. The ligands recognized by γδ TCRs remain in most cases a matter of speculation, but several lines of evidence implicate host molecules elicited by infection or inflammation. In this way a limited set of inducible host molecules might serve as switches that alter immune mechanisms by stimulating the responses of particular γδ T cell subsets. Our findings also suggest that γδ T cell subsets that control inflammatory tissue damage come at a cost: decreased host resistance to a bacterial infection. In this context we are intrigued by observations (56) (A. Mukasa et al., manuscript in preparation) suggesting that certain γδ T cells subsets, during the course of infection with agents such as Mycobacterium tuberculosis and L. monocytogenes, are naturally depleted via a Fas ligand-dependent apoptotic pathway, a normal mechanism that could lead to increased host resistance.

We thank Michael Lahn (National Jewish Medical and Research Center) for careful reading of the manuscript, Bill Townend (National Jewish) for assistance with flow cytometry, and Elizabeth Pflum and Anatole Konoval (National Jewish Medical and Research Center) for technical assistance.

1

This work was supported by National Institutes of Health Grant R01AI44920 (to R.L.O.), a grant from the Rocky Mountain Chapter of the Arthritis Foundation (to R.L.O.), and Environmental Protection Agency/National Jewish Environmental Lung Center Program Project Grant R825793.

3

Abbreviations used in this paper: Ham IgG, normal hamster serum IgG; Vγ4/6−/− mice, gene-targeted mice homozygous for induced mutations inactivating both the Vγ4 and Vγ6 genes; TCRδ−/− mice, gene-targeted mice homozygous for an inactivating mutation in TCR-Cδ; TCRβ−/− mice, gene-targeted mice homozygous for a deletion in the TCR-Cβ locus.

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