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

Abstract

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

FIG. 1

Representative histograms from human patients with cutaneous leishmaniasis. Each group shows the frequency of cytokine-producing cells after 20 h of culture with LACK, SLA, or medium alone in R1, R2, or R3. The histograms demonstrate the frequencies of cells expressing the indicated molecules as detected using antibodies directly conjugated with either PE (y axis) or FITC (x axis) as described in Materials and Methods. The forward- and side-scatter histograms demonstrate the placement of R1 (small lymphocytes), R2 (blast lymphocytes), and R3 (monocytes/macrophages).

FIG. 2

CD4⁺ T cells from patients with cutaneous leishmaniasis express a higher percentage of experienced T cells. The average frequency of CD4⁺ CD45RO⁺ T cells within the CD4⁺-T-cell population is represented for noninfected individuals (n = 6) and patients with cutaneous leishmaniasis (n = 13). The difference was significant using Student's t test with a P value of <0.05.

FIG. 3

Multiple sources of IFN-γ-producing cells in response to SLA, with little LACK-induced IFN-γ. Using single-cell cytoplasmic staining and analysis by flow cytometry, the frequencies of lymphocyte subpopulations producing IFN-γ were determined. The first column represents data obtained from the small lymphocyte gate, R1 (Fig. 1). (A) Percentage of IFN-γ-producing cells in the total lymphocyte population; (B) percentage of CD4⁺ cells producing IFN-γ in the whole population; (C) percentage of cells producing IFN-γ within the CD4⁺-T-cell population. The second column represents data obtained from the lymphocyte blast gate, R2 (as shown in fig. 1). (D) Percentage of IFN-γ⁺-producing cells in the whole gated population; (E) percentage of CD4⁺ IFN-γ-producing T cellsin the whole gated population; (F) the percentage of IFN-γ-producing T cells within the CD4⁺ population; (G) the percentage of CD8⁺ IFN-γ-producing T cells in the whole gated population; (H) percentage of IFN-γ-producing T cells within the CD8⁺ population. Data are the means ± standard errors for 13 individual patients with cutaneous leishmaniasis. The means were compared using the statistical program JMP and Student's t test; comparison of all pairs was made with a P value of <0.05.

FIG. 4

SLA induces a higher frequency of TNF-α-producing lymphocytes than LACK. (A) Percentage of TNF-α-producing cells in R1; (B) percentage of TNF-α-producing cells in the large lymphocyte region, R2. In all cases the frequency of cells after stimulation with SLA was significantly greater than after stimulation with LACK or medium alone. (MED). Data are the means ± standard errors for 13 individual patients with cutaneous leishmaniasis. The frequency of positive cells was determined using intracellular cytoplasmic staining of cytokines in conjunction with cell surface markers and flow cytometry analysis. The means were compared using the statistical program JMP and Student's t test; comparison of all pairs was made with a P value of <0.05.

FIG. 5

The frequencies of IL-10-producing cells are equivalent among the various stimuli. The first column represents data obtained from the small lymphocyte gate, R1 (Fig. 1). (A) Percentage of lymphocytes producing IL-10 in the whole population; (B) percentage of CD4⁺ IL-10⁺-producing T cells in the whole population; (C) percentage of IL-10⁺-producing cells within the CD4⁺ population. The second column represents data obtained from the lymphocyte blast gate, R2 (as shown in figure 1). (D) Percentage of lymphocytes producing IL-10 in the whole population; (E) percentage of CD4⁺ IL-10-producing T cells in the whole population; (F) percentage of IL-10-producing cells within the CD4⁺population. Data are the means ± standard errors for 13 individual patients with cutaneous leishmaniasis. The frequency of positive cells was determined using intracellular cytoplasmic staining of cytokines in conjunction with cell surface markers followed by using flow cytometry analysis. The antibodies used were anti-CD4–FITC and anti-IL-10–PE. The means were compared using the statistical program JMP, and Student's t test; comparison of all pairs was made with a P value of <0.05. In all cases, there were no significant differences among the stimuli.

FIG. 6

Frequency of cytokine-producing cells in the macrophage gate, R3. (A) Percentage of CD14⁺ TNF-α-producing monocytes in the whole gated population; (B) percentage of CD14⁺ IL-12-producing monocytes in the whole gated population; and (C) percentage of IL-10-producing monocytes. The means were compared using the statistical program JMP and Student's t test; comparison of all pairs was made with a P value of <0.05. In all cases, there were no significant differences using the 95% confidence interval.