Antigen specific correlations of cellular immune responses in human leishmaniasis suggests mechanisms for immunoregulation

Abstract

Regulation of the immune response directed against Leishmania is critical for the establishment of effective control of the disease. It is likely that some types of immune responses directed against Leishmania can lead to more severe clinical forms of leishmaniasis causing a poor control of the pathogen and/or pathology, while others lead to resolution of the infection with little pathology as in cutaneous leishmaniasis. To gain a better understanding of the possible role that subpopulations of T cells, and their associated cytokines have on disease progression and/or protective immune responses to L. braziliensis infection, a detailed study of the frequency of activated and memory T cells, as well as antigen specific, cytokine producing T cells was carried out. Following the determination of cytokine producing mononuclear cell populations in response to total Leishmania antigen (SLA), and to the recombinant antigen LACK, correlation analysis were performed between specific cytokine producing populations to identify models for cellular mechanisms of immunoregulation in human cutaneous leishmaniasis. These studies have shown: (1) a positive correlation between ex vivo CD45RO frequencies and antigen specific cytokine (IFN-gamma or IL-10) producing cells; (2) a negative correlation between ex vivo CD69 expression and the frequency of IFN-gamma producing cells; (3) a positive correlation amongst SLA specific, IFN-gamma or TNF-alpha and IL-10 producing lymphocytes with one another; and (4) a higher frequency of IL-10 producing, parasite specific (anti-SLA or anti-LACK), lymphocytes are correlated with a lower frequency of TNF-alpha producing monocytes, demonstrating an antigen specific delivery of IL-10 inducing negative regulation of monocyte activity.

Fig. 1

CD45RO expression is correlated with cytokine producing lymphocytes. Correlation between CD69 or CD45RO expression analysed ex vivo and CD4⁺ T lymphocytes producing IFN-γ stimulated with SLA for 20 h as described in Materials and Methods. (a) Demonstrates the correlation between CD69 and IFN-γ expression (n = 9). (b) Correlation between CD45RO expression analysed ex vivo and production of IFN-γ (n = 9) or (c) IL-10 by SLA specific CD4⁺ T lymphocytes (n = 8). The graphs show fit lines with 95% confidence curves.

Fig. 2

Positive correlation between SLA specific cytokine producing lymphocytes. The frequency of cytokine producing lymphocytes was determined following SLA stimulation of PBMC from cutaneous leishmaniasis patients for 20 h. IL-10 and IFN-γ production (a) by CD4⁺ T lymphocytes and (b) by blast lymphocytes. Total TNF-α and IFN-γ production (c) by lymphocytes and (d) by blast lymphocytes. The graphs show fit lines with 95% confidence curves. (n = 8)

Fig. 3

Positive correlation between TNF-α and IL-10 producing monocytes. Percentage of cytokine-producing monocytes was determined following the indicated culture conditions of PBMC from cutaneous leishmaniasis patients for 20 h. (a) TNF-α and IL-10 production without stimuli (n = 10); (b) stimulated with SLA (n = 9); (c) stimulated with LACK (n = 8), and (d) stimulated with anti-CD3/CD28 (n = 9). The graphs show fit lines with 95% confidence curves.

Fig. 4

Higher frequencies of parasite specific IL-10 producing lymphocytes are correlated with the down modulation of TNF-α producing monocytes. (a) IL-10 production, obtained from the lymphocytes gate versus TNF-α production from monocytes, after culture with SLA (n = 9), and (b) anti-CD3/CD28 (n = 8). (c) IL-10 produced by blast lymphocytes stimulated with LACK versus TNF-α produced by monocytes from cultures stimulated with SLA (n = 9). PBMC from cutaneous leishmaniasis patients were stimulated as indicated for 20 h as described in Materials and Methods. The graphs show fit lines with 95% confidence curves.

Fig. 5

Inhibition of LACK induced IL-10 leads to an increase in TNF-α producing monocytes. PBMC from two cutaneous leishmaniasis patients were stimulated with LACK in the presence or absence of anti-IL-10 mAb for 20 h as described in Materials and Methods. The points on the left side indicate the frequency of TNF-α producing lymphocytes for both individuals (•), or the frequency of TNF-α+/CD14 + cells (▴), or the frequency of total TNF-α producing cells (□). The corresponding points on the right side indicate the same frequencies of TNF-α producing cells from paired cultures in the presence of anti-IL-10. Below are dot plots demonstrating the frequency of TNF-α and CD14 expressing cells for both individuals from cultures with or without neutralizing anti-IL-10 antibodies.