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The Journal of Immunology, 2003, 171: 1447-1455.
Copyright © 2003 by The American Association of Immunologists

Leptospira interrogans Activation of Human Peripheral Blood Mononuclear Cells: Preferential Expansion of TCR{gamma}{delta}+ T Cells vs TCR{alpha}{beta}+ T Cells 1

Gary R. Klimpel2,*, Michael A. Matthias{dagger} and Joseph M. Vinetz*,{dagger}

Departments of * Microbiology and Immunology and {dagger} Pathology, World Health Organization Collaborating Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Innate and adaptive immune responses induced by leptospirosis have not been well characterized. In this study we show that in vitro exposure of naive human PBMC to Leptospira interrogans results in cell proliferation and the production of IFN-{gamma}, IL-12, and TNF-{alpha}. Cell proliferation was highest when using high numbers of Leptospira. Optimal cell proliferation occurred at 6–8 days, and the majority of cells contained in these cultures were {gamma}/{delta} T cells. These cultures showed a 10- to 50-fold expansion of {gamma}/{delta} T cells compared with the initial cellular input. Additionally, these cultures contained elevated numbers of NK cells. In contrast, exposure of PBMC to low numbers of Leptospira failed to induce {gamma}{delta} T cell or NK cell expansion, but induced significant {alpha}{beta} T cell expansion. V{gamma}9/V{delta}2 were expressed on all {gamma}/{delta} T cells expanded by exposure of PBMC to Leptospira. Leptospira stimulation of purified TCR{gamma}{delta}+ T cells, obtained from 8-day cultures of Leptorspira-stimulated PBMC, induced high levels of IFN-{gamma} production, but no cell proliferation, suggesting that such stimulation of {gamma}{delta} T cells did not depend on specialized accessory cells or Ag processing. Finally, in patients with acute leptospirosis, there was a significant (4- to 5-fold) increase in the number of peripheral blood TCR{gamma}{delta}+ T cells. These results indicate that Leptospira can activate {gamma}{delta} T cells and {alpha}{beta} T cells and will guide further investigations into the roles of these T cell populations in host defense and/or the pathology of leptospirosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leptospirosis is a zoonotic disease caused by spirochetes of the genus Leptospira (1). This disease is found worldwide in both temperate and tropical climates (2) and is an important public health threat in the United States as well as abroad (1). There are >200 serologically defined varieties (serovars) of pathogenic Leptospira currently divided into 11 genomospecies (3). Infection by Leptospira spp. can lead to widely divergent clinical outcomes: symptomatic infection, common in endemic regions (4); an undifferentiated febrile illness or an aseptic meningitis syndrome with low morbidity (5); or fulminant disease with a septic shock-like syndrome, jaundice, renal failure, myocarditis/heart failure, hemorrhage, meningitis, and death (3), such as characterizes the ongoing epidemic of severe leptospirosis in urban Brazil (6).

Historically, some leptospiral serovars have been considered to be more virulent than others (hence, serovar designations such as icterohaemorragiae). Yet population-based studies indicate that the relationship between infecting serovar and clinical manifestations of the disease remains far from clear; purportedly virulent serovars can cause mild disease and vice versa (3, 7, 8, 9). It is not clear that antibiotic treatment of leptospirosis leads to improved outcome; clinical studies conflict (10, 11). Therefore, just as in relapsing fever caused by spirochetes of the genus Borrelia (12), it is of substantial importance to delineate mechanisms by which Leptospira activate the immune system so as to point out novel ways to approach the treatment of this possibly fatal illness.

Elevated levels of soluble IL-2R, IL-6, and TNF-{alpha} have been demonstrated in sera obtained from patients treated for acute leptospirosis (13). Recently, heat-killed Leptospira was also shown to induce IFN-{gamma} and IL-12 production from human whole blood cultures (14). Clinical hallmarks of severe leptospirosis can resemble Gram-negative sepsis, with multiorgan failure, refractory hypotension, and death. However, the pathogenic mechanism(s) from either the host or the pathogen side that results in the clinical manifestations of human leptospirosis remains unclear.

Indeed, knowledge of the host immune response to Leptospira or the pathogenesis of leptospirosis remains limited (1). Naturally acquired immunity that protects against reinfection by Leptospira does occur and has been shown to be serovar-specific in animal models (15). It has been assumed that naturally acquired immunity is humorally mediated (15, 16, 17). It has been proposed that immunity is linked to Abs directed against oligosaccharides of serovar-specific leptospiral LPS (17, 18), and that leptospiral LPS stimulation of the innate immune system via a Toll-like receptor 2 (TLR2)3-dependent mechanism may be important in leptospirosis (19). There is also evidence that Abs specific to Leptospira membrane-associated proteins may play a role in host defense (16, 20). The recent observation that high grade bacteremia (101–106/ml) in leptospirosis can occur in the presence of moderate or high titer anti-leptospiral agglutinating Abs makes it plausible that mechanisms other than anti-LPS Abs play a role in naturally acquired protective immunity (21).

The role of cell-mediated immunity in host defense to Leptospira remains poorly understood in both animal models and human disease. Insight into protective humoral and cellular immune responses against Leptospira has been gained from studies of cattle given a killed L. borgpetersenii vaccine. A recent study has shown that cattle immunized with a killed Leptospira vaccine have CD4+ T cells and {gamma}{delta} T cells that give in vitro proliferative responses and produce IFN-{gamma} following stimulation with a Leptospira Ag preparation (22). An uncharacterized leptospiral glycolipoprotein has been shown to induce in vitro production of TNF-{alpha} and IL-10 as well as up-regulate the expression of CD69 and HLA DR on PBMCs (23). In a C3H/HeJ murine model of leptospirosis, it was found that treatment of weanling mice with anti-CD4 mAb seemed to exacerbate morphologically observable pathological damage during infection with a virulent strain of L. interrogans serovar icterohaemorrhagiae (24). Both animal models and human clinical studies have provided indirect evidence that TCR{gamma}{delta}+ T cells may play an important role in host defense against bacterial, viral, and parasitic infections (25, 26, 27, 28, 29, 30, 31, 32). TCR{gamma}{delta}+ T cells have been shown to recognize, independently of Ag processing or APC, nonpeptide phosphoantigens produced by different pathogens (33, 34, 35) and to be capable of producing either Th1 or Th2 cytokines (36, 37). The role that TCR{gamma}{delta}+ T cells might play in human leptospirosis has not been investigated. Additionally, little is known about how human PBMC respond to in vitro exposure to Leptospira. In this report we show that Leptospira induces the in vitro production of Th1 cytokines by PBMC and induces cell proliferation in both {alpha}{beta} T cells and {gamma}{delta} T cells. Finally, {gamma}{delta} T cells were shown to recognize Leptospira without Ag processing or APC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria and reagents

Leptospira interrogans serovar copenhageni (strain M20) were grown in modified Tween 80/albumin liquid culture (PLM-5; Serologicals Corp., Norcross, GA) to log phase by diluting a stationary phase culture 1/20 in fresh PLM-5 and harvesting 48 h later. Bacteria were washed once and adjusted to the correct density in PBS. In some experiments bacteria were heat-killed by exposure to 70°C for 45 min. Abs used for flow cytometric analysis were as follows: FITC- or PE-conjugated mouse mAbs against human CD3, CD4, CD8, TCR{alpha}{beta}, TCR{gamma}{delta}, V{delta}1, V{delta}2, V{gamma}9, and isotype-matched control Abs (Caltag Laboratories, Burlingame, CA) or BD PharMingen (San Diego, CA)). mAb cocktails containing tetrameric mAb complexes as well as other reagents required to purify TCR{gamma}{delta}+ T cells by negative selection were obtained from StemCell Technologies (Vancouver, Canada). Isopentenyl pyrophosphate (IPP) was purchased from Sigma-Aldrich (St. Louis, MO).

PBMC cultures

Peripheral blood from healthy volunteers was obtained and used as a source of PBMC. Donors had no history of Leptospira exposure or previous infection. In some experiments PBMC were obtained from patients presenting with acute, undifferentiated, nonmalarial febrile illness who were diagnosed as having acute leptospirosis. These patients were identified at the Consultorio Febriles (out-patient fever clinic) at the Hospital Apoyo (Iquitos, Peru). Three patients were identified to have anti-leptospiral IgM Abs using the PanBio dipstick test (PanBio InDx, Baltimore, MD) and had no other identifiable cause of their febrile illness. PBMC isolated from these patients and from healthy laboratory field workers were shipped in liquid nitrogen to the University of Texas Medical Branch (Galveston, TX), where they assessed by flow cytometry. PBMC were obtained by Ficoll-Hypaque density gradient centrifugation of either venous blood obtained via Vacutainer collection tubes (15% K3EDTA solution; Terumo Medical, Elkton, MD) or buffy coat preparations (38). PBMC were set up in Linbro 24-well plates (Flow Laboratories, McLean, VA) at 1.6 x 106 cells/well with one of the following: 1) medium only, 2) varying numbers of either heat-killed or live Leptospira, or 3) anti-CD3 (1 µg/ml) or staphylococcal enterotoxin B (SEB; 0.05 µg/ml). Cell cultures were initially incubated in antibiotic-free RPMI 1640 medium (Cell Grow, Mediatech, VA) containing 10% heat-inactivated human AB serum (Gemini Bioproducts, Calabasas, CA). After 6 h at 37°C, 1 ml of AIM-V medium containing kanamycin, penicillin, and streptomycin (100 µg/ml) was added to each well. In some experiments cell proliferation was measured, and for these experiments PBMC were set up as described above, but in 96-well microtiter wells at 2 x 105/well. After varying times in culture, each well was pulsed with 1 µCi of [3H]thymidine (Amersham Pharmacia Biotech, Arlington Heights, IL), and cultured for an additional 16 h. The total incorporated counts per minute of [3H]thymidine were determined by a Packard Direct Matrix {beta} Counter (efficiency of counting, 6%; Packard Instruments, Meriden, CT). Results are expressed as the mean counts per minute of triplicate cultures.

Purification of TCR{gamma}{delta}+ T cells

TCR{gamma}{delta}+ T cells were purified from 8-day cultures containing PBMC plus 5 x 107 Leptospira. Purified TCR{gamma}{delta}+ T cells were obtained by negative selection using a magnetic column system (StemCell Technologies, Vancouver, Canada) performed as previously described (38, 39). TCR{gamma}{delta}+ T cells that were CD4 and CD8 negative were purified using anti-CD4, CD19, CD56, CD16, and anti-CD8 tetrameric mAb complexes (StemCell Technologies) in combination with colloidal magnetic dextran iron beads. Briefly, ~5 x 107 lymphocytes were suspended in 1 ml of PBS/2% FCS. This suspension was then incubated with 50 µl of each of the tetramer complexes on ice for 30 min. After an additional incubation with 60 µl of magnetic colloid on ice for 30 min, the cell suspension was loaded onto a magnetic column for separation by gravity. The purity of the TCR{gamma}{delta}+ T cells collected from the flow-through was assessed by FACS analysis. In some experiments purified TCR{gamma}{delta} T cells were established as short term (10–15 days) cell lines by culture with IL-15 (1000 U/ml) plus IL-2 (100 U/ml).

Cytokine ELISA

Levels of IFN-{gamma}, TNF-{alpha}, and IL-12 in culture supernatants were determined by ELISA as we have previously described (38). The capture mAb and biotinylated detection mAb were purchased from BD PharMingen and used at the concentration recommended by the manufacturer. Recombinant cytokines were used as a standard, from 50,000 to 50 pg/ml. Cytokine levels are expressed as the mean ± SE of duplicate samples.

Flow cytometric analysis

The phenotypes of cells present in different PBMC cultures were determined using standard one- or two-color flow cytometry. Briefly, aliquots of 2 x 105 lymphocytes were incubated with a panel of FITC- or PE-conjugated mAbs specific for CD3, CD4, CD8, CD56, CD16, TCR{alpha}{beta}, or TCR{gamma}{delta} in a 96-well, V-bottom microtiter plate (Dynatech Laboratories, Chantilly, VA). After incubation at 4°C for 30 min, the cultures were washed three times with PBS containing 2% FCS and subsequently fixed with 0.5 mM paraformaldehyde (1%) in PBS. These fixed samples were then analyzed with a FACScan flow cytometer (BD Biosciences, Mountain View, CA). The T lymphocytes expressing the {gamma}{delta} form of TCR were further characterized by FACS analysis with anti-human V{delta}1, V{delta}2, and V{gamma}9 (Endogen, Woburn, MA). Isotype control Abs were included in all flow cytometric analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We initiated a series of experiments to investigate whether Leptospira could activate PBMC obtained from normal healthy volunteers, i.e., specifically whether L. interrogans serovar copenhageni strain M20 induced in vitro cytokine production and/or a proliferative response from PBMC. Varying numbers of live Leptospira were added to PBMC. After 6 h, cultures were treated with penicillin and streptomycin at final concentrations of 100 µg/ml, which completely killed the Leptospira. PBMC were exposed to live Leptospira for 6 h before antibiotic treatment, because we have previously shown that optimal human PBMC responses to Gram-positive and negative bacteria require 6 h of PMBC expose to live bacteria (40). PBMC/Leptospira cultures were then assessed for cytokine production at 48 h, and proliferation was assessed at 3 and 6 days of culture. Positive controls for these experiments included stimulation by LPS (100 ng/ml), anti-CD3 (1 µg/ml), and/or SEB (100 ng/ml). Live leptospires induced a significant proliferative response on day 6 of culture that was dose dependent and could be observed as early as day 3 of culture (Fig. 1). Optimal in vitro proliferation was achieved only with high numbers of bacteria (5 x 107 leptospires/ml); higher doses appeared to be inhibitory. High levels of IFN-{gamma}, TNF-{alpha}, and IL-12 were present in culture supernatants at 48 h of culture (Fig. 2). These results indicate that Leptospira can induce PBMC to proliferate and produce Th1 cytokines. These in vitro responses came from individuals who have had no exposure to this organism.



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  		FIGURE 1. Leptospira-induced proliferation of PBMC. PBMC were cultured in microtiter wells at 2 x 105/well with one of the following: 1) medium only, 2) anti-CD3 (1 µg/ml), 3) SEB (0.05 µg/ml), or 4) varying numbers of L. interrogans. Initially culture medium contained no antibiotics, but antibiotics were added to cultures after 6-h incubation at 37°C. Cell proliferation was assessed at 3 days (A) and 6 days (B) by measuring the amount of [3H]thymidine incorporation during the last 16 h of culture. Results are from one experiment that was representative of five separate experiments.

 


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  		FIGURE 2. Leptospira induces in vitro production of Th1 cytokines from PBMC. PBMC were cultured in microtiter wells at 2 x 105/well with one of the following: 1) medium only, 2) SEB (0.05 µg/ml), 3) E. coli LPS (500 ng/ml; purchased from Sigma-Aldrich), or 4) L. interrogans (5 x 107/ml). Initially culture medium contained no antibiotics, but antibiotics were added to cultures after 6-h incubation at 37°C. Cell-free culture supernatants were pooled from duplicate wells and assessed for IFN-{gamma}, TNF-{alpha}, and IL-12 by ELISA. Results are from one experiment that was representative of four separate experiments.

 
Experiments were performed to identify the PBMC populations that expanded in response to Leptospira stimulation. PBMC cultured in 24-well plates and stimulated with Leptospira were harvested at varying times and phenotyped using flow cytometric analysis. The percentages (Fig. 3A) and numbers (Fig. 3B) of TCR{alpha}{beta}+ vs TCR{gamma}{delta}+ vs NK cells (CD16+, CD56+, CD3-) in cultures at 5, 6, 7, and 8 days after exposure to 5 x 107 live bacteria (antibiotics added at 6 h) were determined. A dramatic increase in TCR{gamma}{delta}+ T cells was observed in these cultures as early as 5 days of culture. Fresh PBMC contained 3–5% TCR{gamma}{delta}+ T cells. After 8 days in culture with Leptospira, almost 70% of the cells in these cultures were TCR{gamma}{delta}+ T cells (Fig. 3A). NK cells were also slightly increased in cultures containing Leptospira. In contrast to the expansion in TCR{gamma}{delta}+ T cells, there was a dramatic loss of TCR{alpha}{beta}+ T cells, from 71% in fresh PBMC to 14% on day 8 after exposure to Leptospira. The actual cell yields from these cultures showed a similar pattern. On day 8 of culture, there was a 15-fold increase in the number of TCR{gamma}{delta}+ T cells compared with the initial cellular input (Fig. 3B). In contrast to this expansion, the absolute number of TCR{alpha}{beta}+ T cells was reduced by 68% in these cultures. NK cells had a 3-fold expansion in cell number compared with the initial cellular input. These results were not dependent upon live bacteria, since heat-killed Leptospira gave similar results (Fig. 4). It should be stressed that in all of the above experiments neither IL-2 nor other cytokines were added to the cultures. Thus, TCR{gamma}{delta}+ T cell expansion and TCR{alpha}{beta}+ T cell loss were due to Leptospira stimulation of PBMC.



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  		FIGURE 3. PBMC exposed to high numbers of Leptospira have a preferential expansion of TCR{gamma}{delta}+ T cells. PBMC were cultured in 24-well plates at 1.6 x 106/well and with 5 x 107 Leptospira. Cells were harvested from cultures on days 5, 6, 7, and 8. Cells were washed, counted, and phenotyped using flow cytometry. NK cells are defined as cells that are CD3-, CD56+, CD16+. Data are expressed as the percentage of cells positive for a particular phenotype (A) or as the actual cell yield per culture (B). Data shown are from one experiment that was representative of five independent experiments.

 


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  		FIGURE 4. TCR{gamma}{delta}+ T cell expansion is not dependent upon live Leptospira. PBMC were set up in culture as described in Fig. 3, except some cultures received heat-killed (70°C, 45 min) Leptospira. Cultures were terminated on day 8, and the cells obtained from each culture were phenotyped by flow cytometry. Data shown are from one experiment that was representative of four independent experiments.

 
Leptospir -induced TCR{gamma}{delta}+ T cell expansion, and the parallel TCR{alpha}{beta}+ T cell reduction was dose dependent. PMBC exposed to lower numbers of Leptospira yielded a very different cell population compared with cultures exposed to high numbers of bacteria. Results from a representative experiment are presented in Fig. 5. Control PBMC cultures, receiving only medium, had few TCR{alpha}{beta}+ T cells (2 x 105), but significant numbers of TCR{alpha}{beta}+ T cells (106) were present in cultures given low numbers (106) of Leptospira. At this concentration of Leptospira (106), low, but significant, cell proliferation was observed in the microtiter culture system (Fig. 1). There were no TCR{gamma}{delta}+ T cells present in cultures that were exposed to low numbers of Leptospira. It appears that high does (>5 x 106) of Leptospira induce TCR{gamma}{delta}+ T cell expansion with a parallel reduction in TCR{alpha}{beta}+ T cells, but lower doses of Leptospiria induce TCR{alpha}{beta}+ T cell expansion without expansion of TCR{gamma}{delta}+ T cells.



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  		FIGURE 5. Preferential expansion of TCR{alpha}{beta}+ vs TCR{gamma}{delta}+ T cells depends upon the number of Leptospira added to PBMC cultures. PBMC were set up in culture as described in Fig. 3, except that varying doses of Leptospira were added to different cultures. Cultures were terminated on day 8, and the cells obtained from each culture were phenotyped by flow cytometry. Data shown are from one experiment that was representative of three independent experiments.

 
Flow cytometric analysis was used to investigate whether TCR{gamma}{delta}+ T cells expanded by Leptospira use V{delta}1 and/or V{delta}2 (V{gamma}9/V{delta}2). Cells obtained from day 8 cultures stimulated by 5 x 107 Leptospira were assessed by flow cytometry using mAbs specific for V{delta}1 vs V{delta}2 vs V{gamma}9. All TCR{gamma}{delta}+ T cells stimulated by Leptospira expressed V{gamma}9/V{delta}2 (Fig. 6) and were CD8- and CD4- (data not shown). Since no studies have been reported regarding Leptospira activation of TCR{gamma}{delta}+ T cells, we investigated whether TCR{gamma}{delta}+ T cells could be directly activated by Leptospira and whether this activation required APC. PBMC were stimulated with 5 x 107 Leptospira and TCR{gamma}{delta}+ T cells purified from 24-well plates on day 8 of culture. TCR{gamma}{delta}+ T cells were purified by negative selection as we have previously described (39) (Fig. 7). Purified TCR{gamma}{delta}+ T cells or TCR{gamma}{delta}+ T cell lines obtained from Leptospira-stimulated PBMC were then assessed for their ability to produce IFN-{gamma} and/or proliferate in response to Leptospira. TCR{gamma}{delta}+ T cells were exposed to one of the following: 1) immobilized anti-CD3 Ab (positive control), 2) IPP (50 µM; positive control), or 3) varying numbers of live Leptospira (antibiotics added at 6 h). After 18 h of culture, cell-free supernatants were obtained and assessed for IFN-{gamma} levels by ELISA, and cell proliferation was measured at 24, 48, and 72 h of culture. Leptospira induced high levels of IFN-{gamma} production from TCR{gamma}{delta}+ T cells (Fig. 8) at levels comparable to those produced by anti-CD3 stimulation. IPP, which has been shown to stimulate TCR{gamma}{delta}+ T cells (32), also induced high levels of IFN-{gamma} production. In contrast, TCR{gamma}{delta}+ T cells failed to proliferate in response to exposure to Leptospira, anti-CD3, or IPP at any of the time points measured over 72 h. (data not presented). Identical results (high IFN-{gamma} production without proliferation) were obtained using short term TCR{gamma}{delta}+ T cell lines (data not presented). IFN {gamma} production was mediated via TCR signaling, since pretreatment of TCR{gamma}{delta}+ cells with soluble mAb specific for CD3 or TCR{gamma}{delta} significantly inhibited Leptospira-induced IFN-{gamma} production (Fig. 9).



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  		FIGURE 6. TCR{gamma}{delta}+ T cells expanded from Leptospira-stimulated PBMC express V{gamma}9 and V{delta}2. PBMC were cultured in 24-well plates at 1.6 x 106/well and with 5 x 107 Leptospira. Cells were harvested from cultures on day 8. Cells were washed, counted, and phenotyped using flow cytometry. Data shown are from one experiment that was representative of three independent experiments.

 


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  		FIGURE 7. Purification of TCR{gamma}{delta}+ T cells by negative selection. PBMC were cultured in 24-well plates at 1.6 x 106/well and with 5 x 107 Leptospira. Cells were harvested from cultures on day 8 and used for purifying TCR{gamma}{delta}+ T cells by negative selection. The histogram shows two-color analysis of T cells obtained by negative selection using a magnetic column system. Data shownare from one experiment that was representative of three independent experiments.

 


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  		FIGURE 8. Leptospira-activation of purified TCR{gamma}{delta}+ T cells. Purified TCR{gamma}{delta}+ T cells were obtained from day 8 cultures of PBMC plus 5 x 107 Leptospira as described in Fig. 7. These TCR{gamma}{delta}+ T cells were then exposed to one of the following: 1) medium only, 2) immobilized anti-CD3, or 3) varying numbers of Leptospira. Culture supernatants were collected at 18 h and assessed for levels of IFN-{gamma} by ELISA. Data shown are from one of two experiments that gave identical results.

 


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  		FIGURE 9. Leptospira activation of purified TCR{gamma}{delta}+ T cells is mediated via the TCR. Purified TCR{gamma}{delta}+ T cells were obtained from day 8 cultures of PBMC plus 5 x 107 Leptospira as described in Fig. 7. These TCR{gamma}{delta}+ T cells were then exposed to one of the following: 1) saline, 2) mAb specific for CD3 (2 µg/ml), or 3) an isotype control Ab. After 45 min at 40 C, cells were washed twice and then exposed to 107 Leptospira as described in Fig. 8. Culture supernatants were collected at 18 h and assessed for levels of IFN-{gamma} by ELISA. Data shown are from one of two experiments that gave identical results.

 
Increased numbers of peripheral blood TCR{gamma}{delta} T cells have been documented in a number of different bacterial infections (25, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55). Our results poise the important question of whether TCR{gamma}{delta}+ T cells are also elevated in the peripheral blood of leptospirosis patients. To begin to address this question, we obtained peripheral blood from leptospirosis patients obtained from Iquitos, Peru, an area where leptospirosis is highly endemic (56, 57). PBMC from three leptospirosis patients and five normal healthy controls were assessed by flow cytometry for the number and percentage of TCR{gamma}{delta}+ T cells. PBMC from normal healthy controls contained 3% TCR{gamma}{delta}+ T cells, which is consistent with previous published findings (58). In contrast, a 4- to 5-fold increase (Fig. 10) in TCR{gamma}{delta}+ T cells was observed in PBMC obtained from the three patients diagnosed with acute leptospirosis.



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  		FIGURE 10. Increased numbers of TCR{gamma}{delta}+ T cells are detected in the peripheral blood of patients with acute leptospirosis. PBMC from three leptospirosis patients and five normal healthy controls were assessed by flow cytometry for the number of peripheral blood TCR{gamma}{delta}+ T cells. Data shown are expressed as the mean percentage of CD3+ T cells expressing TCR{gamma}{delta}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Little is known about the human cellular immune response to Leptospira, either in vitro by PBMC or in vivo. The major findings from this study indicate that in vitro exposure of human PBMC to L. interrogans results in T cell and NK cell expansion and the production of cytokines associated with a Th1-type immune response. We found high levels of IFN-{gamma}, TNF-{alpha}, and IL-12 in PBMC cultures exposed to Leptospira. The production of Th1 cytokines is consistent with clinical studies showing high levels of Th1 cytokines in the serum of patients with severe leptospirosis (59). Recently, Diament et al. (23) showed that a glycoprotein from L. interrogans can induce the production of both TNF-{alpha} and IL-10 from human PBMC and can up-regulate the expression of CD69 and HLA-DR on T cells.

We have extended these previous studies by showing that intact Leptospira can induce Th1 cytokines and T cell proliferation in vitro. The precise biochemical nature of the leptospiral products involved in the induction of these responses is currently under investigation. Leptospires have been shown to contain LPS that differs significantly from LPS of Gram-negative bacteria in both structure and toxigenicity (19, 60). LPS from leptospires has less than 1/1000th the biological effects in vitro and in vivo in comparison with LPS from Gram-negative bacteria (61). It has low pyrogenicity in rabbits, has a very low fatty acid content, does not show immunological cross-reactivity with the lipid A component of LPS, and is ineffective in inducing the local Schwartzman reaction (62). Recently, leptospiral LPS and lipoprotein (LipL32) were shown to mediate signaling via TLR2 (19). In contrast, LPS from Gram-negative bacteria mediates signaling via TLR4. Leptospiral peptidoglycan has also been shown to activate complement, to stimulate phagocytosis by human polymorphonuclear leukocytes, and to activate human endothelial cells for increased adherence by neutrophils (63, 64, 65). Thus, a number of possible components of Leptospira could be responsible for inducing a Th1 cytokine profile and/or T cell proliferation from PBMC. The possibility that TLR could play a role in TCR{gamma}{delta} T cell activation must also be considered. Mokuno et al. (66) have shown that mouse TCR{gamma}{delta} T cells express TLR2 mRNA and that TLR2 appears to be involved in the activation of TCR{gamma}{delta} T cells by E. coli lipid A.

In this study we show that there was a preferential in vitro expansion of TCR{gamma}{delta}+ T cells in PBMC cultures exposed to high numbers of Leptospira. T cells expressing {gamma}- and {delta}-chains of the TCR are a unique population of T cells that are substantially different from TCR{alpha}{beta}-expressing T cells: cell surface markers, limited combinatorial diversity of their TCRs, selective anatomical distribution, and the molecular nature of Ags recognized (67, 68, 69). Most circulating human TCR{gamma}{delta}+ T cells bear V{gamma}9/V{delta}2, while most intestinal TCR{gamma}{delta}+ T cells use V{delta}1 (70, 71). Not surprisingly, we show that V{gamma}9/V{delta}2 are expressed by all TCR{gamma}{delta}+ T cells expanded by Leptospira. Significant increases in the number of peripheral blood V{gamma}9/V{delta}2 T cells have been documented in humans in many bacterial and parasitic diseases, such as tuberculosis and malaria (23, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55). In this study we show that patients with acute leptospirosis have increased numbers of peripheral blood TCR{gamma}{delta}+ T cells. Although we have only had the opportunity to study a limited number of patients, the results suggest that this T cell population is expanded in patients with acute leptospirosis.

Exposure of PBMC to high numbers of Leptospira also resulted in a small, but significant, expansion of NK cells. Whether NK cells and/or TCR{gamma}{delta}+ T cells can recognize Leptospira components and whether this recognition plays any role in their in vitro expansion are unknown. Interestingly, TCR{gamma}{delta}+ T cells expressing V{gamma}9/V{delta}2 recognize nonpeptidic phosphorylated and alkylamine Ags secreted by bacteria in a TCR-dependent, but MHC-unrestricted, manner (72, 73, 74). Mycobacteria (34, 72, 75) and Gram-negative bacteria (76, 77, 78) produce these small, nonprotein, phosphate-bearing Ags. They do not require Ag processing, and only recently have some of their structures been precisely identified (33, 72, 79). E. coli was shown to produce phosphoantigens as a result of the Rohmer metabolic pathway of isoprenoid biosynthesis (76, 78), a pathway used by many pathogenic bacteria. Thus, small, nonprotein, phosphate-containing molecules produced by Leptospira could be one possible candidate for how Leptospira activate TCR{gamma}{delta}+ T cells.

TCR{gamma}{delta}+ T cells, purified from day 8 cultures of PBMC stimulated with Leptospira, produce high levels of IFN-{gamma} following stimulation with Leptospira. For these experiments purified TCR{gamma}{delta}+ T cells were exposed to Leptospira in the absence of accessory cells. These results indicate that Leptospira activation of TCR{gamma}{delta}+ T cells does not require specialized accessory cells or Ag processing. This was further confirmed by using short term (10- to 15-day) TCR{gamma}{delta} T cell lines that gave identical results. Interestingly, no cell proliferation was induced in purified TCR{gamma}{delta} T cells or TCR{gamma}{delta} T cell lines following exposure to Leptospira. These results are consistent with previous studies using bovine and human TCR{gamma}{delta} T cells that also showed that cytokine production and cell proliferation did not necessarily correlate (80, 81). In contrast, activation of TCR{gamma}{delta}+ T cells by Mycobacterium tuberculosis Ags is dependent upon accessory cells, but is non-MHC restricted (42). However, small phosphoantigens of M. tuberculosis appear to be associated with larger carrier molecules that require processing to allow phosphate ligands to traffic from phagosomes to the cell surface for stable expression and presentation. TCR{gamma}{delta}+ T cell activation by purified phosphoantigens has been shown to be accessory cell independent, but cell-cell contact was necessary, with T cell to T cell presentation occurring (82). Recently, pamidronate, an aminobisphosphonate, was shown to activate TCR{gamma}{delta}+ T cells, but required monocytes for presentation (83). This comparison highlights the potentially different mechanisms by which leptospires stimulate TCR{gamma}{delta}+ T cells compared with small, nonpeptidic phosphorylated compounds of mycobacteria, for example. TCR{gamma}{delta}+ T cell recognition of Leptospira, without processing, has significant implications for their role in the innate and adaptive immune response associated with leptospirosis. TCR{gamma}{delta}+ T cells can produce either a Th1 or a Th2 cytokine profile, depending upon the stimulus (37). TCR{gamma}{delta}+ T cells can also produce perforin (84, 85) and granulysin (86), potent antimicrobial proteins (87). Recently, TCR{gamma}{delta}+ T cells were shown to have antibacterial activity that was evident in vivo at a time before TCR{gamma}{delta}+ T cell expansion (88). Activated TCR{gamma}{delta}+ T cells have been shown to stimulate and regulate TCR{alpha}{beta}+ T cell responses (67), and TCR{gamma}{delta}+ T cells can be strongly activated by a number of cytokines or combinations of cytokines (IL-7 (89) and IL-15 (90)). Wang et al. (88) have shown that 1% of TCR{gamma}{delta}+ T cells present in human PBMC produce IFN-{gamma} as early as 2 h following isobutyl amine stimulation, peaking on days 3–4. Further, TCR{gamma}{delta}+ T cells produce more cytokines on a per cell basis than do TCR{alpha}{beta}+ T cells (41). Thus, TCR{gamma}{delta}+ T cells could represent a vital bridge between the innate and adaptive immune responses that would provide a rapid response during the time when Ag-specific TCR{alpha}{beta}+ T cells have not yet been recruited or activated. Bacterial stimulation of TCR{gamma}{delta}+ T cells would also have important consequences, such as prompt release of proinflammatory cytokines and chemokines capable of facilitating the onset of local inflammation. Additionally, this pattern recognition (recognition of phosphorylated small molecules, metabolic products of pathogenic bacteria) by V{gamma}9/V{delta}2 T cells could result in a significant expansion of these cells. There is evidence that in humans and monkeys vaccinated with bacillus Calmette-Guérin that TCR{gamma}{delta}+ T cells can develop a form of memory immune phenotype (30, 91).

In vitro exposure of PBMC to low numbers of Leptospira resulted in a significant expansion of TCR{alpha}{beta}+ T cells. Surprisingly, these cultures had no TCR{gamma}{delta}+ T cells. Why high numbers of Leptospira induce TCR{gamma}{delta}+ T cell expansion while low numbers of Leptospira preferentially induce only TCR{alpha}{beta}+ T cell expansion is unclear. High numbers of Leptospira may be selectively toxic to TCR{alpha}{beta}+ T cells and/or induce apoptosis in these T cells. Another possibility is that TCR{gamma}{delta}+ T cells are inducing apoptosis in TCR{alpha}{beta}+ T cells following exposure to high numbers of Leptospiria. In this regard, TCR{gamma}{delta}+ T cells have been shown to induce apoptosis in CD4+ T cells, as demonstrated using human synovial T cells activated by Borrelia burgdorferi (92) and in a mouse model of coxsackievirus-induced myocarditis (31). In both these studies CD4+ Th2 T cells were targets of TCR{gamma}{delta}+ T cells. Although TCR{gamma}{delta}+ T cells have been proposed to play a role in early innate immune responses, they could also down-modulate the immune response as infection is resolved or possibly as a result of an infection being uncontrolled. In support of this hypothesis, it has been found that in the absence of TCR{gamma}{delta}+ T cells, abnormally large granulomatous responses persist after infection with L. monocytogenes or M. tuberculosis (83, 93), and mice depleted of TCR{gamma}{delta}+ T cells have an exaggerated proinflammatory response to L. monocytogenes infection (93). Other evidence indicates that TCR{gamma}{delta}+ T cells can inhibit various immune functions (65). In a model of bovine tuberculosis, TCR{gamma}{delta}+ T cells inhibited TCR{alpha}{beta}+ T cell responses to Mycobacterium Ags (94). Thus, TCR{gamma}{delta}+ T cells could play a complex role in regulating host defense or the pathology associated with leptospirosis.

The data presented here suggest that PBMCs from leptospira-naive individuals produce proinflammatory cytokines and proliferate in vitro in response to leptospira. Further, we show in preliminary studies that patients with acute leptospirosis have increased numbers of peripheral blood TCR{gamma}{delta}+ T cells. The in vitro expansion of TCR{gamma}{delta}+ vs TCR{alpha}{beta}+ T cells is dependent upon the number of leptospires present in PBMC cultures. It has recently been suggested that the clinical outcome of leptospirosis is associated with the level of leptospiremia (21). The complex interaction of pathogen and host that determines the level of bacteremia may also determine the outcome of downstream activation of different T cell populations (TCR{gamma}{delta}+ vs TCR{alpha}{beta}+), which may ultimately be related to the outcome of infection. Prospective studies are in progress to explore the functional and phenotypic characteristics of T cell populations in human leptospirosis and to determine the relationship between TCR{alpha}{beta}+ vs TCR{gamma}{delta}+ T cell expansion and clinical outcome. Such studies may point toward improved treatment of advanced, severe leptospirosis for which antibiotic treatment may be inadequate.


   Footnotes
 
1 This work was supported by grants from the U.S. Public Health Service, National Institutes of Health (1RO1TW/ES05860); the Sealy Memorial Endowment, University of Texas Medical Branch at Galveston; and the Texas Advanced Technology Program (004952-0019-1999). Back

2 Address correspondence and reprint requests to Dr. Gary R. Klimpel, Department of Microbiology and Immunology, University of Texas Medical Branch, Rt. 1070, 301 University Boulevard, Galveston, TX 77555-1070. Back

3 Abbreviations used in this paper: TLR2, Toll-like receptor 2; IPP, isopentenyl pyrophosphate; SEB, staphylococcal enterotoxin B. Back

Received for publication October 16, 2002. Accepted for publication May 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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