Identification of Two Distinct Subpopulations of Leishmania major-Specific T Helper 2 Cells

Mice.BALB/c mice were purchased from Charles River, and 6- to 12-week-old females were used in all experiments. The animal colonies were screened regularly for mouse pathogens and consistently tested negative. Animal experiments were performed in accordance with institutional and Home Office guidelines.

Infections and monitoring of lesions. L. major LV39 (MRHO/SU/59/P strain) was isolated from skin lesions of infected mice and maintained as described previously (7). Infections with 2 × 10⁶ stationary-phase L. major LV39 promastigotes were performed by subcutaneous injection into one footpad in a final volume of 50 μl. Lesion development was monitored weekly by measuring the increase in footpad thickness with a dial caliper (Kröplin Schnelltaster, Schlüchtern, Germany).

Lymphocyte cultures. (i) Primary stimulation.Spleen and lymph node cells (5 × 10⁶/ml) were stimulated in the presence of 4 × 10⁶ live L. major promastigotes/ml (rendered replication incompetent by UV irradiation) at 37°C in 5% CO₂ in air in 24-well Costar plates in a final volume of 1 ml of complete medium (Dulbecco's modified Eagle medium [Gibco] supplemented with 5% heat-inactivated fetal bovine serum [FBS; Gibco], 216 μg of l-glutamine/ml, 5 × 10⁻⁵ M 2-mercaptoethanol, 10 mM HEPES, 100 U of penicillin ml⁻¹, 100 μg of streptomycin/ml). In the experiments in which lymphoid cells were cultured under Th2 conditions, anti-IFN-γ (XMG1.2 [1]; 3 μg/ml) and recombinant mouse IL-4 (X63Ag8-653 [6]; 200 U/ml) were added to the cultures; in the experiments in which cells were cultured under Th1 conditions, anti-IL-4 (11B11 [21]; 10 μg/ml) and recombinant mouse IL-12 (Genetics Institute; lot no. MRB02894-1; 10 U/ml) were added to the cultures. Cells were cultured for the period of time indicated in the figure legends, and then the culture supernatants were collected and frozen prior to cytokine determination.

(ii) Secondary stimulation.Spleen cells (5 × 10⁶/ml) were stimulated in the presence of 4 × 10⁶ live L. major promastigotes/ml as described above. Six days later, the cells were harvested, a Ficoll gradient was performed, and CD4⁺ T cells were purified by magnetic bead separation with anti-CD4 microbeads (Miltenyi Biotec), according to the protocol provided by the manufacturer. The purity of the CD4⁺ T cells was determined by flow cytometry and was >98%. CD4⁺ T cells were restimulated in the presence of L. major-infected antigen-presenting cells prepared as described previously (8) and incubated for 4 days before the culture supernatants were collected for cytokine determination.

Flow-cytometric analysis.Lymphoid cells were stimulated as described above. Six days later, the cells were harvested, a Ficoll gradient was performed, and CD4⁺ T cells were purified by magnetic bead separation with anti-CD4 microbeads. The detection of intracellular cytokine was performed as described previously (24). Briefly, 1.5 × 10⁶ cells were stimulated with 50 ng of phorbol 12-myristate 13-acetate (PMA; Sigma) and 500 ng of ionomycin (Calbiochem) or, as a control, in the presence of complete medium alone for 4 h, with 10 μg of brefeldin A (Sigma) for the last 2 h. Before surface labeling with anti-CD4 (clone H129.19 or RM4-5 [Pharmingen, San Diego, Calif.]) or anti-T1/ST2 (clone DJ8 [13 or Morwell Diagnostics]), cells were preincubated with 1 μg of rat anti-mouse monoclonal antibody CD32/CD16 (FcγII/III receptor; Pharmingen) to reduce nonspecific binding. Cells were washed, fixed with 2% formaldehyde (Sigma), and permeabilized with 0.5% saponin (Sigma) before the anticytokine antibodies or the isotype controls were added (anti-IL-4, clone BVD4-1D11; anti-IFN-γ, clone XMG1.2; anti-IL-10, clone JES5-16E3; anti-IL-5, clone TRFK5; appropriately labeled rat immunoglobulin G1 [Pharmingen]). The detection of the intracellular cytokines was done with an EPICS XL instrument (Beckman Coulter), and data were analyzed with Beckman Coulter Expo32 software.

Analysis of expression of GATA-3 and T-bet. (i) Isolation of CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells.Spleen cells (5 × 10⁶ ml⁻¹) from BALB/c mice infected for 1 week were stimulated with 4 × 10⁶ live L. major promastigotes ml⁻¹. Six days later, the cells were harvested, a Ficoll gradient was performed, and CD4⁺ T cells were purified by magnetic bead separation with anti-CD4 microbeads (Miltenyi Biotec). CD4⁺ T cells were labeled with anti-T1/ST2, and CD4⁺ T1/ST2⁺ T cells (purity, >99%) and CD4⁺ T1/ST2⁻ T cells (purity, 99.5%) were sorted with an EPICS ELITE ESP cell sorter (Beckman Coulter).

(ii) Reverse transcriptase PCR.Total cellular RNA was extracted from CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells with an RNeasy MiniKit (Qiagen) according to the protocol provided by the manufacturer. Reverse transcription was performed on total RNA (1 μg) by using Moloney murine leukemia virus (Life Technologies) with an oligo(dT) primer (Pharmacia).

Aliquots of cDNA derived from CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells were amplified for the detection of GATA-3, T-bet, and hypoxanthine phosphoribosyltransferase (HPRT) (Table 1).

The amplification program consisted in an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min, and then 72°C for 10 min.

The PCR products for GATA-3, T-bet, and HPRT were visualized by electrophoresis of an ethidium bromide-stained 1% agarose gel. Two controls were run concurrently with the experimental samples: a negative control containing all PCR reagents minus the template and a RNA template lacking the reverse transcription step.

Cytokine profile of CD4⁺ T1/ST2⁻ T cells and CD4⁺ T1/ST2⁺ T cells isolated from L. major-infected BALB/c mice 1 week postinfection.To characterize L. major-specific CD4⁺ T1/ST2⁺ T cells isolated from BALB/c mice, we determined their cytokine profile by intracellular cytokine staining. We first established the kinetics of cytokine detection by CD4⁺ T cells (IL-4, IL-5, IL-10, and IFN-γ) 1, 2, 4, and 6 days after antigenic restimulation in vitro. As presented in Table 2, IL-4, IL-5, IL-10, and IFN-γ were clearly detectable 6 days after in vitro antigenic restimulation. To assess the cytokine profile of Th2 cells, two regions were set up in L. major-specific CD4⁺ T cells; the CD4⁺ T1/ST2⁻ population was gated in A (74.9%), and the CD4⁺ T1/ST2⁺ population was gated in B (14.5%) (Fig. 1A). The expression of T1/ST2 was not altered by PMA, ionomycin, and brefeldin A, as the frequencies of CD4⁺ T cells expressing T1/ST2 before and after the polyclonal restimulation were similar (data not shown). Specificity controls are presented in Table 3. As shown in Fig. 1B, there are almost no CD4⁺ T1/ST2⁺ T cells expressing IFN-γ, compared to the CD4⁺ T1/ST2⁻ T cells (1.4 versus 32.1%, respectively) but a high percentage of the CD4⁺ T1/ST2⁺ T cells express IL-4 (37%). These results indicate that L. major-specific CD4⁺ T cells expressing T1/ST2 are Th2 cells, as they produce IL-4 but not IFN-γ. Surprisingly, another type 2 population was detected among the CD4⁺ T1/ST2⁻ T cells: 8.2% of the CD4⁺ T1/ST2⁻ T cells produce IL-4 but not IFN-γ. The expression of IL-4 was shown to be stable as the addition of IL-4 and anti-IFN-γ (Th2 condition) or IL-12 and anti-IL-4 (Th1 condition) to the culture only marginally altered the frequency of cytokine-expressing CD4⁺ T cells during secondary restimulation (Fig. 1C).

• Open in new tab
• Download powerpoint

FIG. 1.

Expression of IL-4 and IFN-γ in L. major-specific CD4⁺ T cells. BALB/c mice were infected with L. major parasites and, 1 week postinfection, the spleens were harvested and restimulated with antigen. After 6 days, a Ficoll gradient was performed, CD4⁺ T cells were purified by MACS, and restimulated with PMA and ionomycin in the presence of brefeldin A as described in Materials and Methods and labeled with anti-T1/ST2, anti-IL-4, and anti-IFN-γ. Gate A was set up on the T1/ST2⁻ T cells, and gate B was set up on the T1/ST2⁺ T cells (A). The frequencies of cytokine-expressing cells were determined by flow cytometry (B). (C) BALB/c mice were infected with L. major parasites and, 1 week postinfection, the spleens were harvested and restimulated in the presence of L. major alone (neutral conditions), L. major and IL-4 and anti-IFN-γ (Th2 condition), or L. major and IL-12 and anti-IL-4 (Th1 condition). After 6 days, the cells were harvested, a Ficoll gradient was performed, and CD4⁺ T cells were purified by magnetic cell sorting (MACS) and were restimulated with L. major-infected antigen-presenting cells. Four days later, the cells were harvested, a Ficoll gradient was performed, and the CD4⁺ T cells were restimulated with PMA and ionomycin in the presence of brefeldin A as described in Materials and Methods and labeled with anti-IL-4. The frequencies of cytokine-expressing CD4⁺ T cells were determined by flow cytometry. The results of one out of three similar experiments are presented here.

To characterize L. major specific CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells further, the expression of other Th2 cytokines was determined. The presence of two distinct subpopulations of Th2 cells among the CD4⁺ T cells was confirmed by the expression and coexpression of IL-10 and IL-4 by both CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells (Fig. 2A). The similar mean fluorescence intensities of cytokine expression (Fig. 2B) suggest that the amounts of IL-4 and IL-10 produced by cells from the two subpopulations of Th2 cells are similar. The coexpression of IL-4 and IL-5 in both Th2 populations was assessed, and the results are presented in Fig. 3. CD4⁺ T1/ST2⁺ T cells expressed each of these type 2 cytokines individually as well as simultaneously. In contrast, IL-5 could not be detected in the CD4⁺ T1/ST2⁻ T-cell subpopulation.

• Open in new tab
• Download powerpoint

FIG. 2.

Expression and coexpression of IL-4 and IL-10 in CD4⁺ T1/ST2⁺ and CD4⁺ T1/ST2⁻ T cells. (A) BALB/c mice were infected with L. major parasites and, 1 week postinfection, the spleens were harvested and restimulated with antigen. After 6 days, a Ficoll gradient was performed and CD4⁺ T cells were purified by MACS and restimulated with PMA and ionomycin in the presence of brefeldin A as described in Materials and Methods and labeled with anti-T1/ST2, anti-IL-4, and anti-IL-10. Gate A was set up on T1/ST2⁻ T cells, and gate B was set up on T1/ST2⁺ T cells. The frequencies of cytokine-expressing cells were determined by flow cytometry. (B) White histograms, intensity of expression of IL-4 and IL-10 in CD4⁺ T1/ST2⁺ T cells; grey histograms, intensity of expression of IL-4 and IL-10 in CD4⁺ T1/ST2⁻ T cells. Both histograms are scaled to 100% of the peak values. The results of one out of three similar experiments are presented.

• Open in new tab
• Download powerpoint

FIG. 3.

Expression and coexpression of IL-4 and IL-5 in CD4⁺ T1/ST2⁺ and in CD4⁺ T1/ST2⁻ T cells. BALB/c mice were infected with L. major parasites and, 1 week postinfection, the spleens were harvested and restimulated with antigen. After 6 days, a Ficoll gradient was performed, CD4⁺ T cells were purified by MACS and restimulated with PMA and ionomycin in the presence of brefeldin A as described in Materials and Methods and labeled with anti-T1/ST2, anti-IL-4, and anti-IL-5. Gate A was set up on T1/ST2⁻ T cells, and gate B was set up on T1/ST2⁺ T cells. The frequencies of IL-4- and IL-5-expressing cells were determined by flow cytometry. The results of one out of three similar experiments are presented.

The results presented in Fig. 1 to 3 clearly show that CD4⁺ T cells expressing T1/ST2 are Th2 cells. These data also indicate that, after L. major infection, two different populations of Th2 cells are generated in vivo and that they can be distinguished by their expression of the T1/ST2 molecule and IL-5.

Cytokine profile of CD4⁺ T1/ST2⁻ T cells and CD4⁺ T1/ST2⁺ T cells isolated from L. major-infected BALB/c mice 4 weeks postinfection.To determine whether the early cytokine pattern is stable and maintained during the course of cutaneous leishmaniasis, the intracellular cytokine profile of antigen-specific cells was assessed 4 weeks postinfection, a time when the nonhealing Th2 phenotype is completely established, and compared to that obtained 1 week postinfection. As presented in Fig. 4A, populations of IL-4-expressing CD4⁺ T cells were detected in both the CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells derived from cultures from the popliteal lymph node cells (32.5 and 22.0%, respectively). Surprisingly, a small percentage of CD4⁺ T1/ST2⁺ T cells also express IFN-γ (4.8%) or IFN-γ and IL-4 (2.7%). An even higher percentage of Th0-like cells, i.e., CD4⁺ T cells coexpressing IL-4 and IFN-γ, was detected among the CD4⁺ T1/ST2⁻ T-cell population (12.4%).

• Open in new tab
• Download powerpoint

FIG. 4.

Expression and coexpression of Th1 and Th2 cytokines in CD4⁺ T1/ST2⁺ and CD4⁺ T1/T2⁻ T cells. BALB/c mice were infected with L. major parasites and, 4 weeks postinfection, the draining lymph nodes were harvested and restimulated with antigen. After 6 days, a Ficoll gradient was performed, and CD4⁺ T cells were purified by MACS and restimulated with PMA and ionomycin in the presence of brefeldin A as described in Materials and Methods and labeled with anti-T1/ST2, anti-IL-4, and anti-IFN-γ, anti-IL-4 and anti-IL-10, or anti-IL-10 and anti-IL-5. Gate A was set up on the T1/ST2⁻ T cells, and gate B was set up on the T1/ST2⁺ T cells (Fig. 1A). The frequencies of cytokine-expressing cells were determined by flow cytometry. The results of one out of three similar experiments are presented.

CD4⁺ T1/ST2⁺ T cells also express IL-10 alone (7.2%) or in combination with IL-4 (14.7%) (Fig. 4B). Type 2 cells were also present among the CD4⁺ T1/ST2⁻ T cells as shown by the expression of IL-10 (5.3%) or IL-4 and IL-10 (6.8%). Although at a lower frequency, a similar levels of expression or coexpression of IFN-γ, IL-4, or IL-10 were obtained with CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells isolated from the spleen cell cultures (data not shown).

In contrast to the results obtained with the CD4⁺ T1/ST2⁺ T cells from mice infected for 1 week (Fig. 3), intracellular expression of IL-5 was not detectable 4 weeks postinfection (data not shown) and it was low in both the CD4⁺ T1/ST2⁺ T cells (1.6%) and the CD4⁺ T1/ST2⁻ T cells from the lymph node cell cultures (1.1%; Fig. 4C).

In summary, the results presented in Fig. 4 show that two subpopulations of Th2 cells are present in established, nonhealing L. major infections.

Expression of GATA-3 and T-bet in CD4⁺ T1/ST2⁺ and CD4⁺ T1/ST2⁻ T cells.To verify the Th2 nature of the two subpopulations of Th2 cells, the expression of the Th2-specific transcription factor GATA-3 in the CD4⁺ T1/ST2⁺ and the CD4⁺ T1/ST2⁻ T cells was determined. In addition, the expression of T-bet, a Th1-specific transcription factor, was also determined. As shown in Fig. 5, GATA-3 is clearly expressed in the CD4⁺ T1/ST2⁺ T cells (Fig. 5A, lane 2) but T-bet could not be detected (Fig. 5B, lane 2), confirming the Th2 nature of this subset. GATA-3 was also detected in the CD4⁺ T1/ST2⁻ T cells (Fig. 5A, lane 1), further supporting the presence of a distinct population of CD4⁺ T1/ST2⁻ type 2 cells in L. major-infected BALB/c mice.

• Open in new tab
• Download powerpoint

FIG. 5.

Expression of GATA-3 and T-bet in CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells. The expression of GATA-3 (A), T-bet (B), and HPRT (C) in CD4⁺ T1/ST2⁺ T cells and CD4⁺ T1/ST2⁻ T cells was determined by reverse transcriptase PCR as described in Materials and Methods. Lanes 1, CD4⁺ T1/ST2⁻ T cells; lanes 2, CD4⁺ T1/ST2⁺ T cells; lanes 3 to 5, negative controls run with the RNA template but without the reverse transcription step with CD4⁺ T1/ST2⁻ (lanes 3) and CD4⁺ T1/ST2⁺ (lanes 4) T cells and run with all PCR reagents but without the template (lanes 5).