Disparate Immunoregulatory Potentials for Double-Negative (CD4– CD8–)
http://www.100md.com
《感染与免疫杂志》
Department of Biochemistry and Immunology
Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Immunology Service, Hospital Edgard Santos, UFBA, Salvador, Bahia, Brazil
ABSTRACT
Although most T lymphocytes express the T-cell receptor and either CD4 or CD8 molecules, a small population of cells lacking these coreceptors, CD4– CD8– (double negative [DN]) T cells, has been identified in the peripheral immune system of mice and humans. To better understand the role that this population may have in the human immune response against Leishmania spp., a detailed study defining the activation state, cytokine profile, and the heterogeneity of DN T cells bearing or T-cell receptors was performed with a group of well-defined cutaneous leishmaniasis patients. Strikingly, on average 75% of DN T cells from cutaneous leishmaniasis patients expressed the T-cell receptor, with the remainder expressing the receptor, while healthy donors displayed the opposite distribution with 75% of the DN T cells expressing the T-cell receptor. Additionally, DN T cells from cutaneous leishmaniasis patients are compatible with previous antigen exposure and recent activation. Moreover, while DN T cells from Leishmania-infected individuals present a proinflammatory cytokine profile, DN T cells express a regulatory profile exemplified by interleukin-10 production. The balance between these subpopulations could allow for the formation of an effective cellular response while limiting its pathogenic potential.
INTRODUCTION
T lymphocytes recognize antigens as peptides bound to major histocompatibility complex (MHC) molecules. The fine specificity of the T cell is defined by the T-cell receptor (TCR). Most T lymphocytes express the TCR and either CD4 or CD8 molecules (22). These molecules stabilize the TCR-peptide/MHC interaction, are essential for intrathymic selection, and contribute to transmembrane signaling, with important roles in the development and activation of helper and cytotoxic T cells (8, 10, 17). A small population of T cells lacking these coreceptors, CD4– CD8– (double negative [DN]) T cells, have been identified in the peripheral immune system of mice and humans and have been associated with human and murine autoimmune and immunodeficiency diseases (4, 15).
While little is known concerning DN T cells and their direct role in disease pathology or normal immunity in humans, recent studies indicate that the lack of coreceptors allows this population to tolerate chronic stimulation. In contrast, chronic stimulation of CD4 or CD8 T cells limits their expansion through an apoptosis-dependent mechanism (14). In the murine model, DN T cells exhibit markers of activation/memory, a lowered threshold of activation, ex vivo cytolytic activity, and the ability to rapidly secrete gamma interferon (IFN-). Furthermore, they can also compete for interleukin-2 (IL-2) produced by helper T cells, thereby inhibiting the expansion of bystander CD8 T cells, suggesting their role in immunoregulation (25). In healthy humans, DN T cells have been identified in peripheral blood, thymus, and skin (13, 21, 31). This population constitutes a rare population of cells in healthy subjects, expressing molecules associated with activation, memory, and cytotoxic function (6). Another interesting point was reported by Porcelli et al. (24), who showed that proliferative and cytotoxic responses of DN T cells specific for Mycobacterium tuberculosis are restricted to the nonpolymorphic MHC-like CD1 molecules.
Immunity against Leishmania spp. is cell mediated. Usually, this parasite escapes the humoral immune response residing in the phagolysosome of macrophages, which employs a number of defense strategies against the infecting parasite. Lymphocytes also play an essential role in the immune response against Leishmania infection, induced by Th1 clones during infection. IL-12-producing phagocytic cells, as well as IFN- produced by natural killer cells and previously activated T cells, induce the differentiation of IFN-- and tumor necrosis factor alpha (TNF-)-producing T lymphocytes. TNF- produced by the macrophages can also act in an autocrine manner, activating itself for nitric oxide production, which is toxic for the parasite (20, 26). Thus, IFN- is an important cytokine produced in response to Leishmania antigens, leading to an efficient cell-mediated immune response. Recently, data from our laboratory (5) demonstrated that in peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients, DN lymphocytes are the second most abundant source of IFN--producing cells following CD4+ T cells. Moreover, Amprey et al. recently demonstrated the involvement of CD1d-restricted DN T cells in the response to Leishmania in infected mice (1). Thus, to better understand the role DN T cells may have in the human immune response to Leishmania, a detailed study of the activation state and cytokine profiles of both and DN T cells in a group of well-defined CL patients was performed.
MATERIALS AND METHODS
Preparation of the antigens. The soluble Leishmania antigen (SLA) of Leishmania braziliensis was provided by the Leishmaniasis Laboratory (ICB/UFMG/Brazil; W. Mayrink) and is a freeze-thawed antigen preparation. Briefly, L. braziliensis promastigotes (MHOM/BR/81/HJ9) were washed and adjusted to 108 promastigotes/ml in phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO), followed by repeated freeze-thaw cycles and a final ultrasonication. After centrifugation, the supernatant was harvested and the protein concentration was measured by the Lowry method.
Patients and controls. The PBMC analyzed in this study were obtained from two groups of patients studied at different times: 16 patients ranging from 11 to 52 years old (25.62 ± 10.63 years [mean ± standard deviation]) and 9 individuals ranging from 5 to 74 years old (33.75 ± 20.16 years) from Corte de Pedra, Bahia, Brazil, an area where leishmaniasis is endemic due to infection with L. braziliensis. Diagnosis of leishmaniasis was based on clinical findings, a positive skin test for Leishmania antigens, and/or positive parasitological exams. All presented with one or two ulcerated lesions between 15 days and 3 months of duration (Table 1). None of the individuals reported prior infections with Leishmania. All individuals participated in the study through informed consent and received treatment whether or not they chose to participate in the study. PBMC were also obtained from a group of six healthy donors, with ages ranging from 20 to 27 years old (23.33 ± 2.73 years). These studies were approved by the National Ethical Clearance Committee of Brazil as well as local ethical committee clearances and abide by the Helsinki Declaration on human subject research.
In vitro cultures. All cultures were carried out using RPMI 1640 supplemented with 5% AB Rh-positive heat-inactivated human serum, antibiotics (200 U/ml penicillin), and 1 mM L-glutamine (Sigma Chemical Co., St. Louis, MO), in the absence or presence of SLA (10 μg/ml).
Staining to determine lymphocyte profile and single-cell cytoplasmic cytokine staining. PBMC were obtained by separating whole blood over Ficoll (Sigma Chemical Co., St. Louis, MO), stained, and analyzed for their profile and intracellular cytokine expression pattern as described before (5). Briefly, 2.5 x 105 PBMC were analyzed ex vivo or after being cultured in 96-well plates in 200-μl cultures for 20 h with either medium alone or SLA (final concentration of 10 μg/ml). PBMC were incubated with biotinylated (biot) fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, cy-chrome (Cy)-, or tricolor (TRI)-labeled antibody solutions for 20 min at 4°C. After being washed, the wells with biotin were incubated with 20 μl of FITC- or Cy-labeled streptavidin (1:100) solutions for 20 min at 4°C. Then, the preparations were fixed with 200 μl of 2% formaldehyde (Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline and acquired using a fluorescence-activated cell sorter (FACS) (FACSCalibur; Becton Dickinson, San Jose, CA) or stained for cytoplasmic cytokine. In the last case, during the last 4 h of culture, brefeldin A (1 μg/ml) was added to the cultures before the surface markers were stained. The fixed cells were permeabilized with a solution of saponin, stained using anticytokine monoclonal antibodies, fixed, and analyzed using FACS. At least 35,000 gated events were acquired for later analysis.
The antibodies used for staining were immunoglobulin FITC and PE controls, anti-CD3-FITC, anti-CD4-FITC, anti-CD8-FITC, anti-CD28-FITC, anti-CD69-FITC, anti-CD95-FITC, anti--FITC, anti--FITC, anti-CD69-PE, anti-CD25-PE, anti-CD45RO-PE, anti-CD28-PE, anti-IFN--PE, anti-TNF--PE, anti-IL-10-PE, anti-CD4-TRI, anti-CD8-TRI, (Caltag, Carlsbad, CA), anti-CD4-biot, anti-CD8-biot, anti-CD3-PE, anti-CD25-PE, anti-IFN--PE, anti-TNF--PE, anti-IL-10-PE, anti-IL-4-PE, and anti-CD56-Cy (PharMingen, San Diego, CA).
Analysis of FACS data. Lymphocytes were analyzed using the program Cell Quest (Becton Dickinson, San Jose, CA). The use of certain combinations of antibodies allowed the separation of two and, in several cases, three lymphocyte populations, CD4, CD8, and DN T lymphocytes or DN lymphocytes and DN lymphocytes. Three different fluorochromes were associated for each analysis. In the first case, anti-CD4-biot-SA-Cy, anti-CD8-biot-SA-Cy, anti-CD3-FITC or PE, and a fourth molecule, a surface marker or a cytokine conjugated with PE or FITC, were used. In this manner, for example, the regions of CD3+ (FITC) and CD4– and CD8– (biot-SA-Cy) were selected and another region was generated for analysis of the third fluorochrome. In the second case, anti-CD4-TRI, anti-CD8-TRI, anti--FITC or anti--FITC, and a fourth molecule, a surface marker or a cytokine conjugated with PE, were used. Statistical analysis was performed using the Student t test or Wilcoxon test contained in JMP, a statistical program from SAS.
RESULTS
Clinical features. The clinical profile of the 25 CL patients used in this study is shown in Table 1. The duration of lesions ranged from 15 to 90 days (48.04 ± 26.91 days) at the time the blood was taken and measurements were made. The total area of the ulcers varied from 16 to 2,691 mm2 (499.72 ± 597.83 mm2). All patients presented with positive Leishmania skin tests (Montenegro skin test), while measurements for 20 patients ranged from 35 to 439 mm2 (224.85 ± 108.82 mm2).
The DN T-cell population from CL patients is highly activated and skewed toward the production of inflammatory cytokines compared to that of noninfected controls. We previously demonstrated that DN lymphocytes are the second most abundant source of IFN--producing cells in PBMC from CL patients (5). To gain a better understanding of the role this population may have in the formation of protective or pathogenic immune responses in human disease, we first compared the phenotypic characteristics of the entire DN T-cell population (containing both and DN T cells) from CL patients with those from noninfected individuals.
The frequencies of DN T cells from healthy and Leishmania-infected individuals ranged from 3.77% to 6.92% (mean, 5.85%) and 0.59% to 4.95% (mean, 2.94%), respectively, as determined using flow cytometry. The frequencies of ex vivo DN T cells expressing the activation markers CD69 and HLA-DR were higher in leishmaniasis patients than in noninfected controls (Table 2). Moreover, the frequencies of DN T cells expressing the costimulatory molecule CD28 and the memory marker CD45RO were also higher in Leishmania-infected patients than in noninfected controls (Table 2). After culture in medium alone, the expressions of CD69, CD56, and cytokine production were analyzed for the entire DN T-cell population. Again, the DN T cells from leishmaniasis patients showed higher frequencies of CD69- and CD56-expressing cells. Moreover, the frequencies of IFN-- and TNF--producing cells were also increased; however, the frequency of IL-4-producing cells was equivalent (Table 3).
The DN T-cell population from CL patients is skewed toward TCR-expressing cells which display increased activation and inflammatory potential both ex vivo and after culture in media alone. After determining that the DN T-cell subpopulation from CL patients, as a whole, displays a hyperactivated profile compared to that of noninfected controls, we undertook an analysis of the and TCR-expressing DN T cells separately both ex vivo and after in vitro culture with media alone.
The and DN T-cell subpopulations were first analyzed ex vivo. The proportion of and DN T cells from noninfected individuals ranged from 0.92% to 2.30% (mean, 1.22%) and 2.12% to 6.83% (mean, 3.17%), respectively. Thus, an average of 27% of the DN T cells express the TCR in noninfected individuals (Fig. 1A). In contrast, the proportion of DN and DN cells from CL patients ranged from 1.87% to 5.28% (mean, 3.30%) (Fig. 1B) and 0.33% to 1.75% (mean, 1.27%) (Fig. 1B), respectively, with the DN T cells making up an average of 72% of the DN T cells in CL patients.
Next, the activation states of the and DN T-cell populations were determined for both CL patients and healthy individuals by determining the frequency of cells expressing CD25, CD28, CD69, and CD45RO within each subpopulation (Table 4). Similar to that found with the whole DN T-cell population in CL patients, the DN T-cell population expressed an ex vivo hyperactivated state, measured by CD69 expression, compared to the DN T cells from noninfected individuals (Table 4). Additionally, CL patients expressed a high frequency of CD28+ DN T cells compared to healthy controls. However, the frequencies of and DN T cells expressing CD25, and CD45RO were not different when analyzed ex vivo (Table 4).
Spontaneous in vitro culture can allow for further activation and cytokine production in several cell populations; thus, cultures were performed in the absence of external stimuli by using media alone. Again, as with the whole DN T-cell population, the individual subpopulations from CL patients displayed a hyperactivated profile compared to those of the noninfected individuals (Table 4). Both the and DN T cells expressed higher frequencies of inflammatory cytokines, such as IFN- and TNF-, and the anti-inflammatory cytokine IL-10.
DN T cells from CL patients express an exacerbated inflammatory cytokine profile, while the DN T cells express a regulatory cytokine profile. To better understand the role that the and DN T cells may have in mounting of protective and/or pathogenic immune responses in human leishmaniasis, we compared the SLA-induced cytokine production pattern of each subpopulation from CL patients. In all cases, an antigen-induced change was seen following SLA culture of cells from CL patients. Importantly, no induction of activation markers, nor of cytokine production, was seen for the and DN T cells from noninfected individuals (data not shown).
Sharp differences were seen in the relative frequencies of cytokine-expressing cells when the and DN T-cell subpopulations were compared. DN T cells displayed higher frequencies of inflammatory cytokine-producing cells than DN T cells. The frequency of IFN--producing cells within the DN T cells was higher than that seen in DN cells in spontaneous cultures (Table 4); however, as seen in Fig. 2A, this difference was amplified following culture with SLA (mean, 19.69% versus 10.95% for / versus /, respectively). Moreover, the frequency of TNF--producing cells within the population (mean, 8.21%) was 5.6 times higher than that seen with DN T cells (mean, 1.15%) when the cultures were performed with SLA (Fig. 2B). In contrast, the frequency of DN T cells producing the regulatory cytokine IL-10 (6.46%) was sevenfold higher than that seen with the DN T-cell subpopulation (0.92%) following stimulation with SLA (Fig. 2C). Interestingly, this difference was seen only after antigen-specific culture as no difference was seen in cultures performed with medium alone (Table 4).
An important measure of immunoregulatory capacity of a given cell population is the balance between inflammatory and regulatory cytokine-producing antigen-specific cells, and thus, individual inflammatory ratios, percent IFN-/percent IL-10 and percent TNF-/percent IL-10, were calculated for each subpopulation of DN T cells. Strikingly, the IFN-- and TNF--based ratios for the DN T cells were 20.78 ± 15.89 and 10.41 ± 9.27 while the same ratios for the DN T cells were 1.64 ± 0.85 and 0.18 ± 0.15, respectively.
DISCUSSION
The complexity of leishmaniasis is created through the interaction between a range of parasite strains with a heterogenic host immune response. Despite the complex range of diseases and responses associated with this disease, several cytokines and their cellular sources have been correlated with the cure for and/or pathology of leishmaniasis. One critical cytokine that is produced by several T-lymphocyte subpopulations is IFN-. While CD4+ Th1 cells are responsible for the majority of IFN- production, the second most prevalent source present in peripheral blood of CL patients was identified as CD4– CD8– lymphocytes (5).
In this report, we establish that this DN lymphocyte population from CL patients is, in fact, composed of a DN T-cell population that expresses a hyperactivated and inflammatory profile compared to DN T cells from noninfected individuals. While this is present in a relative minority compared to other T-cell populations, its highly activated state makes it likely important in the overall immune response against Leishmania as was recently demonstrated in a murine model of leishmaniasis (1). In our hands, the mean frequency of DN T cells from healthy controls was higher than that observed for Leishmania-infected patients. This decrease could be accounted for by differential recruitment of this population to the lesion site and/or draining lymph nodes. In fact, correlation analysis shows that the larger the lesion area, the lower the frequency of DN T cells in the peripheral blood (data not shown). Further studies by our group are under way to determine the frequency and nature of DN T cells in patient lesions.
Using TCR transgenic mice, Caveno and coworkers demonstrated that the stimulation and the optimal proliferative response of DN T cells is dependent on the interaction of CD28 and B7 (9). Although a lower expression of CD28 is associated with cell activation in many cases (22a), we observed that a high frequency of CD28-negative cells was not observed within the DN subpopulation. It is possible that the interaction of CD28 with its ligands plays an important role in activation of the DN T cells, since they do not express CD4 or CD8, and thus is not down-modulated upon activation. In the present study, DN T cells from infected patients expressed increased levels not only of this costimulatory molecule but also of recent activation markers and memory molecules ex vivo. The increased expression of CD69 in infected patients is sustained after short-term culture. Another characteristic of DN T cells, observed here and described before (6), is the high frequency of cells expressing CD56, which is associated with cytotoxicity activity. Thus, overall, the DN T population from CL patients presented a profile compatible with previous antigen exposure, recent activation, and cytotoxic function.
The cure for leishmaniasis is related to the presence of a strong Th1 response and memory, leading to the production of IFN- and TNF- which activate parasite-infected macrophages for parasite destruction (11, 12). DN T cells displayed a high commitment to the production of IFN-, as already observed by our group, but they also contained a higher frequency of cells producing another Th1 cytokine, TNF-. This biased profile of high TNF- and IFN-, with a lower frequency of IL-4 and IL-10, will favor the activity of DN T cells as inducers of cell-mediated immunity through the activation of phagocytes and the induction of Th1 differentiation through the modulation of microenvironments. Since the development of pathogenic responses in human leishmaniasis is related to an inability to modulate type 1 immune responses (9a), DN T cells, if not well regulated, might also aid in the development of immunopathology. In fact, correlation analysis showed that the higher the frequency of IFN--producing lymphocytes, the larger the lesion size (2).
Studies of human leishmaniasis have suggested the participation of subsets contributing to the host immune response during infection. The majority of infiltrating T cells in cutaneous, mucosal, and diffuse forms of leishmaniasis express the TCR, while cells were abundant only in the cutaneous form of the disease (30). However, upon Leishmania antigen stimulation, the expansion of peripheral cells in vivo and in vitro was seen in several clinical forms. Although cells proliferate in response to promastigote lysate, two immunodominant antigens, gp63 and gp42, that elicit strong cell proliferation, did not trigger cell expansion, suggesting a different pattern of antigen recognition by these subsets (7, 28, 29). In contrast, in the murine model, CD4+, CD8+, or CD4– CD8– T cells from vaccinated or infected donors conferred significant disease resistance when they were transferred to nave recipients and then infected with Leishmania mexicana (19).
While approximately 75% of the DN cells from Leishmania-infected patients studied here bear the TCR on their surface, approximately 25% of this population bears the TCR type. However, in healthy individuals, the opposite is seen, with the cells being the predominant subpopulation, comprising 72.21% of the DN cells. Given the importance of further investigation to precisely identify the specific role of versus DN cell subpopulations, we studied them separately. In addition to representing the majority among the DN T cells in PBMC from Leishmania-infected patients, the DN cells were also identified as being the major proinflammatory cytokine-expressing cells and were capable of producing both IFN- and TNF-. Interestingly, following stimulation with SLA, the anti-inflammatory potential of DN cells rose dramatically and fell within the subpopulation. The opposite effect was seen with respect to TNF- production, which was increased within the subpopulation and decreased within the DN cells. Thus, in Leishmania-infected patients, the inflammatory component resides within the DN T-cell subpopulation, while the regulatory component resides within the DN T-cell subpopulation.
Recent studies have shown that T cells from Leishmania donovani-infected patients exhibited a high frequency of IFN- and TNF- coexpression. However, this subpopulation was also highly committed toward IL-10 production, suggesting a modulatory role of T cells during visceral leishmaniasis (18). IL-10 has been associated with susceptibility to L. donovani infection in mice and humans (16, 23) but has also been related to prevention of pathology due to exacerbated Th1 responses triggered by the Leishmania during the cutaneous form of the disease (3). Although it has been shown that the T cells are expanded within PBMC from patients presenting several forms of the disease and in lesions of mice and humans (27, 29), the precise role of this subpopulation in leishmaniasis is still not clear.
In conclusion, these studies demonstrate fundamental differences between the two subpopulations that make up the DN T cells from Leishmania-infected patients. We found that the DN cells present an increased bias in their capacity to induce inflammatory immune responses and that the DN subpopulation shows a regulatory profile. Since the inflammatory response is not only essential for control of the parasite burden but also responsible for tissue injury, we suggest that both subpopulations are important during the establishment of an efficient protective immune response. The DN population would activate macrophages, leading to increased antiparasitic capacity, while the DN population may act in more of a regulatory role to modulate the inflammation. Together, our results suggest a role for DN T cells in the induction of cell-mediated immunity and possibly in immunopathology if unregulated.
Further investigation to precisely identify which antigens and presenting molecules are responsible for the activation of the DN T cells from human leishmaniasis and to determine which DN T cells are involved in the induction of protective versus pathogenic immune responses in different clinical forms of human leishmaniasis will be helpful in understanding the mechanisms involved in the induction of immunity and the regulation of pathology in leishmaniasis.
ACKNOWLEDGMENTS
We acknowledge Wilson Mayrink for the Leishmania antigen and Edgar M. Carvalho for reviewing the manuscript. Walderez O. Dutra and Kenneth J. Gollob are CNPq fellows.
We received financial support from UNICEF/UNDP/WHO-TDR, NIH grants AI-066253 and TMRC AI-30639, NIH training grant TW007127-01, PRONEX-FAPEMIG, FINEP CT-Infra, and CNPq.
FOOTNOTES
Corresponding author. Mailing address: UFMG-ICB, Av. Antonio Carlos, 6627 Belo Horizonte, MG, Brazil. Phone: 55-31-3499-2655. Fax: 55-31-3499-2655. E-mail: kjgollob@gmail.com.
Published ahead of print on 21 August 2006.
REFERENCES
1. Amprey, J. L., G. F. Spth, and S. A. Porcelli. 2004. Inhibition of CD1 expression in human dendritic cells during intracellular infection with Leishmania donovani. Infect. Immun. 72:589-592.
2. Antonelli, L. R., W. O. Dutra, R. P. Almeida, O. Bacellar, E. M. Carvalho, and K. J. Gollob. 2005. Activated inflammatory T cells correlate with lesion size in human cutaneous leishmaniasis. Immunol. Lett. 101:226-230.
3. Antonelli, L. R., W. O. Dutra, R. P. Almeida, O. Bacellar, and K. J. Gollob. 2004. Antigen specific correlations of cellular immune responses in human leishmaniasis suggests mechanisms for immunoregulation. Clin. Exp. Immunol. 136:341-348.
4. Bleesing, J. J., M. R. Brown, J. K. Dale, S. E. Straus, M. J. Lenardo, J. M. Puck, T. P. Atkinson, and T. A. Fleisher. 2001. TcR-alpha/beta(+) CD4(–)CD8(–) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis. Clin. Immunol. 100:314-324.
5. Bottrel, R. L., W. O. Dutra, F. A. Martins, B. Gontijo, E. Carvalho, M. Barral-Netto, A. Barral, R. P. Almeida, W. Mayrink, R. Locksley, et al. 2001. Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble leishmania antigen in human cutaneous leishmaniasis. Infect. Immun. 69:3232-3239.
6. Brooks, E. G., S. P. Balk, K. Aupeix, M. Colonna, J. L. Strominger, and V. Groh-Spies. 1993. Human T-cell receptor (TCR) alpha/beta + CD4–CD8– T cells express oligoclonal TCRs, share junctional motifs across TCR V beta-gene families, and phenotypically resemble memory T cells. Proc. Natl. Acad. Sci. USA 90:11787-11791.
7. Burns, J. M., Jr., J. M. Scott, E. M. Carvalho, D. M. Russo, C. J. March, K. P. Van Ness, and S. G. Reed. 1991. Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses. J. Immunol. 146:742-748.
8. Cammarota, G., A. Scheirle, B. Takacs, D. M. Doran, R. Knorr, W. Bannwarth, J. Guardiola, and F. Sinigaglia. 1992. Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules. Nature 356:799-801.
9. Caveno, J., Y. Zhang, B. Motyka, S. J. Teh, and H. S. Teh. 1999. Functional similarity and differences between selection-independent CD4–CD8– alphabeta T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4–CD8– alphabeta T cells in antigen-expressing mice. J. Immunol. 163:1222-1229.
9a. Faria, D. R., K. J. Gollob, J. Barbosa, Jr., A. Schriefer, P. R. L. Machado, H. Lessa, L. P. Carvalho, M. A. Romano-Silva, A. R. de Jesus, E. M. Carvalho, and W. O. Dutra. 2005. Decreased in situ expression of interleukin-10 receptor is correlated with the exacerbated inflammatory and cytotoxic responses observed in mucosal leishmaniasis. Infect. Immun. 73:7853-7859.
10. Gay, D., C. Coeshott, W. Golde, J. Kappler, and P. Marrack. 1986. The major histocompatibility complex-restricted antigen receptor on T cells. IX. Role of accessory molecules in recognition of antigen plus isolated IA. J. Immunol. 136:2026-2032.
11. Gollob, K. J., L. R. Antonelli, and W. O. Dutra. 2005. Insights into CD4+ memory T cells following Leishmania infection. Trends Parasitol. 21:347-350.
12. Green, S. J., R. M. Crawford, J. T. Hockmeyer, M. S. Meltzer, and C. A. Nacy. 1990. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha. J. Immunol. 145:4290-4297.
13. Groh, V., M. Fabbi, F. Hochstenbach, R. T. Maziarz, and J. L. Strominger. 1989. Double-negative (CD4–CD8–) lymphocytes bearing T-cell receptor alpha and beta chains in normal human skin. Proc. Natl. Acad. Sci. USA 86:5059-5063.
14. Hamad, A. R., A. Srikrishnan, P. Mirmonsef, C. P. Broeren, C. H. June, D. Pardoll, and J. P. Schneck. 2001. Lack of coreceptor allows survival of chronically stimulated double-negative alpha/beta T cells: implications for autoimmunity. J. Exp. Med. 193:1113-1121.
15. Illum, N., E. Ralfkiaer, G. Pallesen, and C. Geisler. 1991. Phenotypical and functional characterization of double-negative (CD4–CD8–) alpha beta T-cell receptor positive cells from an immunodeficient patient. Scand. J. Immunol. 34:635-645.
16. Kenney, R. T., D. L. Sacks, A. A. Gam, H. W. Murray, and S. Sundar. 1998. Splenic cytokine responses in Indian kala-azar before and after treatment. J. Infect. Dis. 177:815-818.
17. Konig, R., L. Y. Huang, and R. N. Germain. 1992. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature 356:796-798.
18. Lagler, H., M. Willheim, F. Traunmuller, K. Wahl, H. Winkler, M. Ramharter, W. Graninger, and S. Winkler. 2003. Cellular profile of cytokine production in a patient with visceral leishmaniasis: gammadelta+ T cells express both type 1 cytokines and interleukin-10. Scand. J. Immunol. 57:291-295.
19. Lezama-Davila, C. M., and G. Gallagher. 1995. CD4+, CD8+ and CD4– CD8– T cell-subsets can confer protection against Leishmania m. mexicana infection. Mem. Inst. Oswaldo Cruz 90:51-58.
20. Liew, F. Y., D. Xu, and W. L. Chan. 1999. Immune effector mechanism in parasitic infections. Immunol. Lett. 65:101-104.
21. Londei, M., A. Verhoef, B. P. De, M. Kissonerghis, B. Grubeck-Loebenstein, and M. Feldmann. 1989. Definition of a population of CD4–8– T cells that express the alpha beta T-cell receptor and respond to interleukins 2, 3, and 4. Proc. Natl. Acad. Sci. USA 86:8502-8506.
22. Marrack, P., C. Hannum, M. Harris, K. Haskins, R. Kubo, M. Pigeon, R. Shimonkevitz, J. White, and J. Kappler. 1983. Antigen-specific, major histocompatibility complex-restricted T cell receptors. Immunol. Rev. 76:131-145.
22a. Menezes, C. A., M. O. Rocha, P. E. Souza, A. C. Chaves, K. J. Gollob, and W. O. Dutra. 2004. Phenotypic and functional characteristics of CD28+ and CD28– cells from chagasic patients: distinct repertoire and cytokine repression. Clin. Exp. Immunol. 137:129-138.
23. Murphy, M. L., U. Wille, E. N. Villegas, C. A. Hunter, and J. P. Farrell. 2001. IL-10 mediates susceptibility to Leishmania donovani infection. Eur. J. Immunol. 31:2848-2856.
24. Porcelli, S., C. T. Morita, and M. B. Brenner. 1992. CD1b restricts the response of human CD4–8– T lymphocytes to a microbial antigen. Nature 360:593-597.
25. Priatel, J. J., O. Utting, and H. S. Teh. 2001. TCR/self-antigen interactions drive double-negative T cell peripheral expansion and differentiation into suppressor cells. J. Immunol. 167:6188-6194.
26. Rogers, K. A., G. K. DeKrey, M. L. Mbow, R. D. Gillespie, C. I. Brodskyn, and R. G. Titus. 2002. Type 1 and type 2 responses to Leishmania major. FEMS Microbiol. Lett. 209:1-7.
27. Rosat, J. P., H. R. MacDonald, and J. A. Louis. 1993. A role for gamma delta + T cells during experimental infection of mice with Leishmania major. J. Immunol. 150:550-555.
28. Russo, D. M., J. M. Burns, Jr., E. M. Carvalho, R. J. Armitage, K. H. Grabstein, L. L. Button, W. R. McMaster, and S. G. Reed. 1991. Human T cell responses to gp63, a surface antigen of Leishmania. J. Immunol. 147:3575-3580.
29. Russo, D. M., R. J. Armitage, M. Barral-Netto, A. Barral, K. H. Grabstein, and S. G. Reed. 1993. Antigen-reactive gamma delta T cells in human leishmaniasis. J. Immunol. 151:3712-3718.
30. Tapia, F. J., G. Caceres-Dittmar, M. A. Sanchez, A. E. Fernandez, and J. Convit. 1993. The cutaneous lesion in American leishmaniasis: leukocyte subsets, cellular interaction and cytokine production. Biol. Res. 26:239-247.
31. Toribio, M. L., A. de la Hera, J. R. Regueiro, C. Marquez, M. A. Marcos, R. Bragado, A. Arnaiz-Villena, and C. Martinez. 1988. Alpha/beta heterodimeric T-cell receptor expression early in thymocyte differentiation. J. Mol. Cell. Immunol. 3:347-362.(Lis R. V. Antonelli, Walderez O. Dutra, )
Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Immunology Service, Hospital Edgard Santos, UFBA, Salvador, Bahia, Brazil
ABSTRACT
Although most T lymphocytes express the T-cell receptor and either CD4 or CD8 molecules, a small population of cells lacking these coreceptors, CD4– CD8– (double negative [DN]) T cells, has been identified in the peripheral immune system of mice and humans. To better understand the role that this population may have in the human immune response against Leishmania spp., a detailed study defining the activation state, cytokine profile, and the heterogeneity of DN T cells bearing or T-cell receptors was performed with a group of well-defined cutaneous leishmaniasis patients. Strikingly, on average 75% of DN T cells from cutaneous leishmaniasis patients expressed the T-cell receptor, with the remainder expressing the receptor, while healthy donors displayed the opposite distribution with 75% of the DN T cells expressing the T-cell receptor. Additionally, DN T cells from cutaneous leishmaniasis patients are compatible with previous antigen exposure and recent activation. Moreover, while DN T cells from Leishmania-infected individuals present a proinflammatory cytokine profile, DN T cells express a regulatory profile exemplified by interleukin-10 production. The balance between these subpopulations could allow for the formation of an effective cellular response while limiting its pathogenic potential.
INTRODUCTION
T lymphocytes recognize antigens as peptides bound to major histocompatibility complex (MHC) molecules. The fine specificity of the T cell is defined by the T-cell receptor (TCR). Most T lymphocytes express the TCR and either CD4 or CD8 molecules (22). These molecules stabilize the TCR-peptide/MHC interaction, are essential for intrathymic selection, and contribute to transmembrane signaling, with important roles in the development and activation of helper and cytotoxic T cells (8, 10, 17). A small population of T cells lacking these coreceptors, CD4– CD8– (double negative [DN]) T cells, have been identified in the peripheral immune system of mice and humans and have been associated with human and murine autoimmune and immunodeficiency diseases (4, 15).
While little is known concerning DN T cells and their direct role in disease pathology or normal immunity in humans, recent studies indicate that the lack of coreceptors allows this population to tolerate chronic stimulation. In contrast, chronic stimulation of CD4 or CD8 T cells limits their expansion through an apoptosis-dependent mechanism (14). In the murine model, DN T cells exhibit markers of activation/memory, a lowered threshold of activation, ex vivo cytolytic activity, and the ability to rapidly secrete gamma interferon (IFN-). Furthermore, they can also compete for interleukin-2 (IL-2) produced by helper T cells, thereby inhibiting the expansion of bystander CD8 T cells, suggesting their role in immunoregulation (25). In healthy humans, DN T cells have been identified in peripheral blood, thymus, and skin (13, 21, 31). This population constitutes a rare population of cells in healthy subjects, expressing molecules associated with activation, memory, and cytotoxic function (6). Another interesting point was reported by Porcelli et al. (24), who showed that proliferative and cytotoxic responses of DN T cells specific for Mycobacterium tuberculosis are restricted to the nonpolymorphic MHC-like CD1 molecules.
Immunity against Leishmania spp. is cell mediated. Usually, this parasite escapes the humoral immune response residing in the phagolysosome of macrophages, which employs a number of defense strategies against the infecting parasite. Lymphocytes also play an essential role in the immune response against Leishmania infection, induced by Th1 clones during infection. IL-12-producing phagocytic cells, as well as IFN- produced by natural killer cells and previously activated T cells, induce the differentiation of IFN-- and tumor necrosis factor alpha (TNF-)-producing T lymphocytes. TNF- produced by the macrophages can also act in an autocrine manner, activating itself for nitric oxide production, which is toxic for the parasite (20, 26). Thus, IFN- is an important cytokine produced in response to Leishmania antigens, leading to an efficient cell-mediated immune response. Recently, data from our laboratory (5) demonstrated that in peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients, DN lymphocytes are the second most abundant source of IFN--producing cells following CD4+ T cells. Moreover, Amprey et al. recently demonstrated the involvement of CD1d-restricted DN T cells in the response to Leishmania in infected mice (1). Thus, to better understand the role DN T cells may have in the human immune response to Leishmania, a detailed study of the activation state and cytokine profiles of both and DN T cells in a group of well-defined CL patients was performed.
MATERIALS AND METHODS
Preparation of the antigens. The soluble Leishmania antigen (SLA) of Leishmania braziliensis was provided by the Leishmaniasis Laboratory (ICB/UFMG/Brazil; W. Mayrink) and is a freeze-thawed antigen preparation. Briefly, L. braziliensis promastigotes (MHOM/BR/81/HJ9) were washed and adjusted to 108 promastigotes/ml in phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO), followed by repeated freeze-thaw cycles and a final ultrasonication. After centrifugation, the supernatant was harvested and the protein concentration was measured by the Lowry method.
Patients and controls. The PBMC analyzed in this study were obtained from two groups of patients studied at different times: 16 patients ranging from 11 to 52 years old (25.62 ± 10.63 years [mean ± standard deviation]) and 9 individuals ranging from 5 to 74 years old (33.75 ± 20.16 years) from Corte de Pedra, Bahia, Brazil, an area where leishmaniasis is endemic due to infection with L. braziliensis. Diagnosis of leishmaniasis was based on clinical findings, a positive skin test for Leishmania antigens, and/or positive parasitological exams. All presented with one or two ulcerated lesions between 15 days and 3 months of duration (Table 1). None of the individuals reported prior infections with Leishmania. All individuals participated in the study through informed consent and received treatment whether or not they chose to participate in the study. PBMC were also obtained from a group of six healthy donors, with ages ranging from 20 to 27 years old (23.33 ± 2.73 years). These studies were approved by the National Ethical Clearance Committee of Brazil as well as local ethical committee clearances and abide by the Helsinki Declaration on human subject research.
In vitro cultures. All cultures were carried out using RPMI 1640 supplemented with 5% AB Rh-positive heat-inactivated human serum, antibiotics (200 U/ml penicillin), and 1 mM L-glutamine (Sigma Chemical Co., St. Louis, MO), in the absence or presence of SLA (10 μg/ml).
Staining to determine lymphocyte profile and single-cell cytoplasmic cytokine staining. PBMC were obtained by separating whole blood over Ficoll (Sigma Chemical Co., St. Louis, MO), stained, and analyzed for their profile and intracellular cytokine expression pattern as described before (5). Briefly, 2.5 x 105 PBMC were analyzed ex vivo or after being cultured in 96-well plates in 200-μl cultures for 20 h with either medium alone or SLA (final concentration of 10 μg/ml). PBMC were incubated with biotinylated (biot) fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, cy-chrome (Cy)-, or tricolor (TRI)-labeled antibody solutions for 20 min at 4°C. After being washed, the wells with biotin were incubated with 20 μl of FITC- or Cy-labeled streptavidin (1:100) solutions for 20 min at 4°C. Then, the preparations were fixed with 200 μl of 2% formaldehyde (Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline and acquired using a fluorescence-activated cell sorter (FACS) (FACSCalibur; Becton Dickinson, San Jose, CA) or stained for cytoplasmic cytokine. In the last case, during the last 4 h of culture, brefeldin A (1 μg/ml) was added to the cultures before the surface markers were stained. The fixed cells were permeabilized with a solution of saponin, stained using anticytokine monoclonal antibodies, fixed, and analyzed using FACS. At least 35,000 gated events were acquired for later analysis.
The antibodies used for staining were immunoglobulin FITC and PE controls, anti-CD3-FITC, anti-CD4-FITC, anti-CD8-FITC, anti-CD28-FITC, anti-CD69-FITC, anti-CD95-FITC, anti--FITC, anti--FITC, anti-CD69-PE, anti-CD25-PE, anti-CD45RO-PE, anti-CD28-PE, anti-IFN--PE, anti-TNF--PE, anti-IL-10-PE, anti-CD4-TRI, anti-CD8-TRI, (Caltag, Carlsbad, CA), anti-CD4-biot, anti-CD8-biot, anti-CD3-PE, anti-CD25-PE, anti-IFN--PE, anti-TNF--PE, anti-IL-10-PE, anti-IL-4-PE, and anti-CD56-Cy (PharMingen, San Diego, CA).
Analysis of FACS data. Lymphocytes were analyzed using the program Cell Quest (Becton Dickinson, San Jose, CA). The use of certain combinations of antibodies allowed the separation of two and, in several cases, three lymphocyte populations, CD4, CD8, and DN T lymphocytes or DN lymphocytes and DN lymphocytes. Three different fluorochromes were associated for each analysis. In the first case, anti-CD4-biot-SA-Cy, anti-CD8-biot-SA-Cy, anti-CD3-FITC or PE, and a fourth molecule, a surface marker or a cytokine conjugated with PE or FITC, were used. In this manner, for example, the regions of CD3+ (FITC) and CD4– and CD8– (biot-SA-Cy) were selected and another region was generated for analysis of the third fluorochrome. In the second case, anti-CD4-TRI, anti-CD8-TRI, anti--FITC or anti--FITC, and a fourth molecule, a surface marker or a cytokine conjugated with PE, were used. Statistical analysis was performed using the Student t test or Wilcoxon test contained in JMP, a statistical program from SAS.
RESULTS
Clinical features. The clinical profile of the 25 CL patients used in this study is shown in Table 1. The duration of lesions ranged from 15 to 90 days (48.04 ± 26.91 days) at the time the blood was taken and measurements were made. The total area of the ulcers varied from 16 to 2,691 mm2 (499.72 ± 597.83 mm2). All patients presented with positive Leishmania skin tests (Montenegro skin test), while measurements for 20 patients ranged from 35 to 439 mm2 (224.85 ± 108.82 mm2).
The DN T-cell population from CL patients is highly activated and skewed toward the production of inflammatory cytokines compared to that of noninfected controls. We previously demonstrated that DN lymphocytes are the second most abundant source of IFN--producing cells in PBMC from CL patients (5). To gain a better understanding of the role this population may have in the formation of protective or pathogenic immune responses in human disease, we first compared the phenotypic characteristics of the entire DN T-cell population (containing both and DN T cells) from CL patients with those from noninfected individuals.
The frequencies of DN T cells from healthy and Leishmania-infected individuals ranged from 3.77% to 6.92% (mean, 5.85%) and 0.59% to 4.95% (mean, 2.94%), respectively, as determined using flow cytometry. The frequencies of ex vivo DN T cells expressing the activation markers CD69 and HLA-DR were higher in leishmaniasis patients than in noninfected controls (Table 2). Moreover, the frequencies of DN T cells expressing the costimulatory molecule CD28 and the memory marker CD45RO were also higher in Leishmania-infected patients than in noninfected controls (Table 2). After culture in medium alone, the expressions of CD69, CD56, and cytokine production were analyzed for the entire DN T-cell population. Again, the DN T cells from leishmaniasis patients showed higher frequencies of CD69- and CD56-expressing cells. Moreover, the frequencies of IFN-- and TNF--producing cells were also increased; however, the frequency of IL-4-producing cells was equivalent (Table 3).
The DN T-cell population from CL patients is skewed toward TCR-expressing cells which display increased activation and inflammatory potential both ex vivo and after culture in media alone. After determining that the DN T-cell subpopulation from CL patients, as a whole, displays a hyperactivated profile compared to that of noninfected controls, we undertook an analysis of the and TCR-expressing DN T cells separately both ex vivo and after in vitro culture with media alone.
The and DN T-cell subpopulations were first analyzed ex vivo. The proportion of and DN T cells from noninfected individuals ranged from 0.92% to 2.30% (mean, 1.22%) and 2.12% to 6.83% (mean, 3.17%), respectively. Thus, an average of 27% of the DN T cells express the TCR in noninfected individuals (Fig. 1A). In contrast, the proportion of DN and DN cells from CL patients ranged from 1.87% to 5.28% (mean, 3.30%) (Fig. 1B) and 0.33% to 1.75% (mean, 1.27%) (Fig. 1B), respectively, with the DN T cells making up an average of 72% of the DN T cells in CL patients.
Next, the activation states of the and DN T-cell populations were determined for both CL patients and healthy individuals by determining the frequency of cells expressing CD25, CD28, CD69, and CD45RO within each subpopulation (Table 4). Similar to that found with the whole DN T-cell population in CL patients, the DN T-cell population expressed an ex vivo hyperactivated state, measured by CD69 expression, compared to the DN T cells from noninfected individuals (Table 4). Additionally, CL patients expressed a high frequency of CD28+ DN T cells compared to healthy controls. However, the frequencies of and DN T cells expressing CD25, and CD45RO were not different when analyzed ex vivo (Table 4).
Spontaneous in vitro culture can allow for further activation and cytokine production in several cell populations; thus, cultures were performed in the absence of external stimuli by using media alone. Again, as with the whole DN T-cell population, the individual subpopulations from CL patients displayed a hyperactivated profile compared to those of the noninfected individuals (Table 4). Both the and DN T cells expressed higher frequencies of inflammatory cytokines, such as IFN- and TNF-, and the anti-inflammatory cytokine IL-10.
DN T cells from CL patients express an exacerbated inflammatory cytokine profile, while the DN T cells express a regulatory cytokine profile. To better understand the role that the and DN T cells may have in mounting of protective and/or pathogenic immune responses in human leishmaniasis, we compared the SLA-induced cytokine production pattern of each subpopulation from CL patients. In all cases, an antigen-induced change was seen following SLA culture of cells from CL patients. Importantly, no induction of activation markers, nor of cytokine production, was seen for the and DN T cells from noninfected individuals (data not shown).
Sharp differences were seen in the relative frequencies of cytokine-expressing cells when the and DN T-cell subpopulations were compared. DN T cells displayed higher frequencies of inflammatory cytokine-producing cells than DN T cells. The frequency of IFN--producing cells within the DN T cells was higher than that seen in DN cells in spontaneous cultures (Table 4); however, as seen in Fig. 2A, this difference was amplified following culture with SLA (mean, 19.69% versus 10.95% for / versus /, respectively). Moreover, the frequency of TNF--producing cells within the population (mean, 8.21%) was 5.6 times higher than that seen with DN T cells (mean, 1.15%) when the cultures were performed with SLA (Fig. 2B). In contrast, the frequency of DN T cells producing the regulatory cytokine IL-10 (6.46%) was sevenfold higher than that seen with the DN T-cell subpopulation (0.92%) following stimulation with SLA (Fig. 2C). Interestingly, this difference was seen only after antigen-specific culture as no difference was seen in cultures performed with medium alone (Table 4).
An important measure of immunoregulatory capacity of a given cell population is the balance between inflammatory and regulatory cytokine-producing antigen-specific cells, and thus, individual inflammatory ratios, percent IFN-/percent IL-10 and percent TNF-/percent IL-10, were calculated for each subpopulation of DN T cells. Strikingly, the IFN-- and TNF--based ratios for the DN T cells were 20.78 ± 15.89 and 10.41 ± 9.27 while the same ratios for the DN T cells were 1.64 ± 0.85 and 0.18 ± 0.15, respectively.
DISCUSSION
The complexity of leishmaniasis is created through the interaction between a range of parasite strains with a heterogenic host immune response. Despite the complex range of diseases and responses associated with this disease, several cytokines and their cellular sources have been correlated with the cure for and/or pathology of leishmaniasis. One critical cytokine that is produced by several T-lymphocyte subpopulations is IFN-. While CD4+ Th1 cells are responsible for the majority of IFN- production, the second most prevalent source present in peripheral blood of CL patients was identified as CD4– CD8– lymphocytes (5).
In this report, we establish that this DN lymphocyte population from CL patients is, in fact, composed of a DN T-cell population that expresses a hyperactivated and inflammatory profile compared to DN T cells from noninfected individuals. While this is present in a relative minority compared to other T-cell populations, its highly activated state makes it likely important in the overall immune response against Leishmania as was recently demonstrated in a murine model of leishmaniasis (1). In our hands, the mean frequency of DN T cells from healthy controls was higher than that observed for Leishmania-infected patients. This decrease could be accounted for by differential recruitment of this population to the lesion site and/or draining lymph nodes. In fact, correlation analysis shows that the larger the lesion area, the lower the frequency of DN T cells in the peripheral blood (data not shown). Further studies by our group are under way to determine the frequency and nature of DN T cells in patient lesions.
Using TCR transgenic mice, Caveno and coworkers demonstrated that the stimulation and the optimal proliferative response of DN T cells is dependent on the interaction of CD28 and B7 (9). Although a lower expression of CD28 is associated with cell activation in many cases (22a), we observed that a high frequency of CD28-negative cells was not observed within the DN subpopulation. It is possible that the interaction of CD28 with its ligands plays an important role in activation of the DN T cells, since they do not express CD4 or CD8, and thus is not down-modulated upon activation. In the present study, DN T cells from infected patients expressed increased levels not only of this costimulatory molecule but also of recent activation markers and memory molecules ex vivo. The increased expression of CD69 in infected patients is sustained after short-term culture. Another characteristic of DN T cells, observed here and described before (6), is the high frequency of cells expressing CD56, which is associated with cytotoxicity activity. Thus, overall, the DN T population from CL patients presented a profile compatible with previous antigen exposure, recent activation, and cytotoxic function.
The cure for leishmaniasis is related to the presence of a strong Th1 response and memory, leading to the production of IFN- and TNF- which activate parasite-infected macrophages for parasite destruction (11, 12). DN T cells displayed a high commitment to the production of IFN-, as already observed by our group, but they also contained a higher frequency of cells producing another Th1 cytokine, TNF-. This biased profile of high TNF- and IFN-, with a lower frequency of IL-4 and IL-10, will favor the activity of DN T cells as inducers of cell-mediated immunity through the activation of phagocytes and the induction of Th1 differentiation through the modulation of microenvironments. Since the development of pathogenic responses in human leishmaniasis is related to an inability to modulate type 1 immune responses (9a), DN T cells, if not well regulated, might also aid in the development of immunopathology. In fact, correlation analysis showed that the higher the frequency of IFN--producing lymphocytes, the larger the lesion size (2).
Studies of human leishmaniasis have suggested the participation of subsets contributing to the host immune response during infection. The majority of infiltrating T cells in cutaneous, mucosal, and diffuse forms of leishmaniasis express the TCR, while cells were abundant only in the cutaneous form of the disease (30). However, upon Leishmania antigen stimulation, the expansion of peripheral cells in vivo and in vitro was seen in several clinical forms. Although cells proliferate in response to promastigote lysate, two immunodominant antigens, gp63 and gp42, that elicit strong cell proliferation, did not trigger cell expansion, suggesting a different pattern of antigen recognition by these subsets (7, 28, 29). In contrast, in the murine model, CD4+, CD8+, or CD4– CD8– T cells from vaccinated or infected donors conferred significant disease resistance when they were transferred to nave recipients and then infected with Leishmania mexicana (19).
While approximately 75% of the DN cells from Leishmania-infected patients studied here bear the TCR on their surface, approximately 25% of this population bears the TCR type. However, in healthy individuals, the opposite is seen, with the cells being the predominant subpopulation, comprising 72.21% of the DN cells. Given the importance of further investigation to precisely identify the specific role of versus DN cell subpopulations, we studied them separately. In addition to representing the majority among the DN T cells in PBMC from Leishmania-infected patients, the DN cells were also identified as being the major proinflammatory cytokine-expressing cells and were capable of producing both IFN- and TNF-. Interestingly, following stimulation with SLA, the anti-inflammatory potential of DN cells rose dramatically and fell within the subpopulation. The opposite effect was seen with respect to TNF- production, which was increased within the subpopulation and decreased within the DN cells. Thus, in Leishmania-infected patients, the inflammatory component resides within the DN T-cell subpopulation, while the regulatory component resides within the DN T-cell subpopulation.
Recent studies have shown that T cells from Leishmania donovani-infected patients exhibited a high frequency of IFN- and TNF- coexpression. However, this subpopulation was also highly committed toward IL-10 production, suggesting a modulatory role of T cells during visceral leishmaniasis (18). IL-10 has been associated with susceptibility to L. donovani infection in mice and humans (16, 23) but has also been related to prevention of pathology due to exacerbated Th1 responses triggered by the Leishmania during the cutaneous form of the disease (3). Although it has been shown that the T cells are expanded within PBMC from patients presenting several forms of the disease and in lesions of mice and humans (27, 29), the precise role of this subpopulation in leishmaniasis is still not clear.
In conclusion, these studies demonstrate fundamental differences between the two subpopulations that make up the DN T cells from Leishmania-infected patients. We found that the DN cells present an increased bias in their capacity to induce inflammatory immune responses and that the DN subpopulation shows a regulatory profile. Since the inflammatory response is not only essential for control of the parasite burden but also responsible for tissue injury, we suggest that both subpopulations are important during the establishment of an efficient protective immune response. The DN population would activate macrophages, leading to increased antiparasitic capacity, while the DN population may act in more of a regulatory role to modulate the inflammation. Together, our results suggest a role for DN T cells in the induction of cell-mediated immunity and possibly in immunopathology if unregulated.
Further investigation to precisely identify which antigens and presenting molecules are responsible for the activation of the DN T cells from human leishmaniasis and to determine which DN T cells are involved in the induction of protective versus pathogenic immune responses in different clinical forms of human leishmaniasis will be helpful in understanding the mechanisms involved in the induction of immunity and the regulation of pathology in leishmaniasis.
ACKNOWLEDGMENTS
We acknowledge Wilson Mayrink for the Leishmania antigen and Edgar M. Carvalho for reviewing the manuscript. Walderez O. Dutra and Kenneth J. Gollob are CNPq fellows.
We received financial support from UNICEF/UNDP/WHO-TDR, NIH grants AI-066253 and TMRC AI-30639, NIH training grant TW007127-01, PRONEX-FAPEMIG, FINEP CT-Infra, and CNPq.
FOOTNOTES
Corresponding author. Mailing address: UFMG-ICB, Av. Antonio Carlos, 6627 Belo Horizonte, MG, Brazil. Phone: 55-31-3499-2655. Fax: 55-31-3499-2655. E-mail: kjgollob@gmail.com.
Published ahead of print on 21 August 2006.
REFERENCES
1. Amprey, J. L., G. F. Spth, and S. A. Porcelli. 2004. Inhibition of CD1 expression in human dendritic cells during intracellular infection with Leishmania donovani. Infect. Immun. 72:589-592.
2. Antonelli, L. R., W. O. Dutra, R. P. Almeida, O. Bacellar, E. M. Carvalho, and K. J. Gollob. 2005. Activated inflammatory T cells correlate with lesion size in human cutaneous leishmaniasis. Immunol. Lett. 101:226-230.
3. Antonelli, L. R., W. O. Dutra, R. P. Almeida, O. Bacellar, and K. J. Gollob. 2004. Antigen specific correlations of cellular immune responses in human leishmaniasis suggests mechanisms for immunoregulation. Clin. Exp. Immunol. 136:341-348.
4. Bleesing, J. J., M. R. Brown, J. K. Dale, S. E. Straus, M. J. Lenardo, J. M. Puck, T. P. Atkinson, and T. A. Fleisher. 2001. TcR-alpha/beta(+) CD4(–)CD8(–) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis. Clin. Immunol. 100:314-324.
5. Bottrel, R. L., W. O. Dutra, F. A. Martins, B. Gontijo, E. Carvalho, M. Barral-Netto, A. Barral, R. P. Almeida, W. Mayrink, R. Locksley, et al. 2001. Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble leishmania antigen in human cutaneous leishmaniasis. Infect. Immun. 69:3232-3239.
6. Brooks, E. G., S. P. Balk, K. Aupeix, M. Colonna, J. L. Strominger, and V. Groh-Spies. 1993. Human T-cell receptor (TCR) alpha/beta + CD4–CD8– T cells express oligoclonal TCRs, share junctional motifs across TCR V beta-gene families, and phenotypically resemble memory T cells. Proc. Natl. Acad. Sci. USA 90:11787-11791.
7. Burns, J. M., Jr., J. M. Scott, E. M. Carvalho, D. M. Russo, C. J. March, K. P. Van Ness, and S. G. Reed. 1991. Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses. J. Immunol. 146:742-748.
8. Cammarota, G., A. Scheirle, B. Takacs, D. M. Doran, R. Knorr, W. Bannwarth, J. Guardiola, and F. Sinigaglia. 1992. Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules. Nature 356:799-801.
9. Caveno, J., Y. Zhang, B. Motyka, S. J. Teh, and H. S. Teh. 1999. Functional similarity and differences between selection-independent CD4–CD8– alphabeta T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4–CD8– alphabeta T cells in antigen-expressing mice. J. Immunol. 163:1222-1229.
9a. Faria, D. R., K. J. Gollob, J. Barbosa, Jr., A. Schriefer, P. R. L. Machado, H. Lessa, L. P. Carvalho, M. A. Romano-Silva, A. R. de Jesus, E. M. Carvalho, and W. O. Dutra. 2005. Decreased in situ expression of interleukin-10 receptor is correlated with the exacerbated inflammatory and cytotoxic responses observed in mucosal leishmaniasis. Infect. Immun. 73:7853-7859.
10. Gay, D., C. Coeshott, W. Golde, J. Kappler, and P. Marrack. 1986. The major histocompatibility complex-restricted antigen receptor on T cells. IX. Role of accessory molecules in recognition of antigen plus isolated IA. J. Immunol. 136:2026-2032.
11. Gollob, K. J., L. R. Antonelli, and W. O. Dutra. 2005. Insights into CD4+ memory T cells following Leishmania infection. Trends Parasitol. 21:347-350.
12. Green, S. J., R. M. Crawford, J. T. Hockmeyer, M. S. Meltzer, and C. A. Nacy. 1990. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha. J. Immunol. 145:4290-4297.
13. Groh, V., M. Fabbi, F. Hochstenbach, R. T. Maziarz, and J. L. Strominger. 1989. Double-negative (CD4–CD8–) lymphocytes bearing T-cell receptor alpha and beta chains in normal human skin. Proc. Natl. Acad. Sci. USA 86:5059-5063.
14. Hamad, A. R., A. Srikrishnan, P. Mirmonsef, C. P. Broeren, C. H. June, D. Pardoll, and J. P. Schneck. 2001. Lack of coreceptor allows survival of chronically stimulated double-negative alpha/beta T cells: implications for autoimmunity. J. Exp. Med. 193:1113-1121.
15. Illum, N., E. Ralfkiaer, G. Pallesen, and C. Geisler. 1991. Phenotypical and functional characterization of double-negative (CD4–CD8–) alpha beta T-cell receptor positive cells from an immunodeficient patient. Scand. J. Immunol. 34:635-645.
16. Kenney, R. T., D. L. Sacks, A. A. Gam, H. W. Murray, and S. Sundar. 1998. Splenic cytokine responses in Indian kala-azar before and after treatment. J. Infect. Dis. 177:815-818.
17. Konig, R., L. Y. Huang, and R. N. Germain. 1992. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature 356:796-798.
18. Lagler, H., M. Willheim, F. Traunmuller, K. Wahl, H. Winkler, M. Ramharter, W. Graninger, and S. Winkler. 2003. Cellular profile of cytokine production in a patient with visceral leishmaniasis: gammadelta+ T cells express both type 1 cytokines and interleukin-10. Scand. J. Immunol. 57:291-295.
19. Lezama-Davila, C. M., and G. Gallagher. 1995. CD4+, CD8+ and CD4– CD8– T cell-subsets can confer protection against Leishmania m. mexicana infection. Mem. Inst. Oswaldo Cruz 90:51-58.
20. Liew, F. Y., D. Xu, and W. L. Chan. 1999. Immune effector mechanism in parasitic infections. Immunol. Lett. 65:101-104.
21. Londei, M., A. Verhoef, B. P. De, M. Kissonerghis, B. Grubeck-Loebenstein, and M. Feldmann. 1989. Definition of a population of CD4–8– T cells that express the alpha beta T-cell receptor and respond to interleukins 2, 3, and 4. Proc. Natl. Acad. Sci. USA 86:8502-8506.
22. Marrack, P., C. Hannum, M. Harris, K. Haskins, R. Kubo, M. Pigeon, R. Shimonkevitz, J. White, and J. Kappler. 1983. Antigen-specific, major histocompatibility complex-restricted T cell receptors. Immunol. Rev. 76:131-145.
22a. Menezes, C. A., M. O. Rocha, P. E. Souza, A. C. Chaves, K. J. Gollob, and W. O. Dutra. 2004. Phenotypic and functional characteristics of CD28+ and CD28– cells from chagasic patients: distinct repertoire and cytokine repression. Clin. Exp. Immunol. 137:129-138.
23. Murphy, M. L., U. Wille, E. N. Villegas, C. A. Hunter, and J. P. Farrell. 2001. IL-10 mediates susceptibility to Leishmania donovani infection. Eur. J. Immunol. 31:2848-2856.
24. Porcelli, S., C. T. Morita, and M. B. Brenner. 1992. CD1b restricts the response of human CD4–8– T lymphocytes to a microbial antigen. Nature 360:593-597.
25. Priatel, J. J., O. Utting, and H. S. Teh. 2001. TCR/self-antigen interactions drive double-negative T cell peripheral expansion and differentiation into suppressor cells. J. Immunol. 167:6188-6194.
26. Rogers, K. A., G. K. DeKrey, M. L. Mbow, R. D. Gillespie, C. I. Brodskyn, and R. G. Titus. 2002. Type 1 and type 2 responses to Leishmania major. FEMS Microbiol. Lett. 209:1-7.
27. Rosat, J. P., H. R. MacDonald, and J. A. Louis. 1993. A role for gamma delta + T cells during experimental infection of mice with Leishmania major. J. Immunol. 150:550-555.
28. Russo, D. M., J. M. Burns, Jr., E. M. Carvalho, R. J. Armitage, K. H. Grabstein, L. L. Button, W. R. McMaster, and S. G. Reed. 1991. Human T cell responses to gp63, a surface antigen of Leishmania. J. Immunol. 147:3575-3580.
29. Russo, D. M., R. J. Armitage, M. Barral-Netto, A. Barral, K. H. Grabstein, and S. G. Reed. 1993. Antigen-reactive gamma delta T cells in human leishmaniasis. J. Immunol. 151:3712-3718.
30. Tapia, F. J., G. Caceres-Dittmar, M. A. Sanchez, A. E. Fernandez, and J. Convit. 1993. The cutaneous lesion in American leishmaniasis: leukocyte subsets, cellular interaction and cytokine production. Biol. Res. 26:239-247.
31. Toribio, M. L., A. de la Hera, J. R. Regueiro, C. Marquez, M. A. Marcos, R. Bragado, A. Arnaiz-Villena, and C. Martinez. 1988. Alpha/beta heterodimeric T-cell receptor expression early in thymocyte differentiation. J. Mol. Cell. Immunol. 3:347-362.(Lis R. V. Antonelli, Walderez O. Dutra, )