Lack of demonstrable memory T cell responses in humans who have spontaneously recovered from leptospirosis in the Peruvian Amazon

Abstract

BACKGROUND. We tested the hypothesis that patients who have recovered from leptospirosis have peripheral blood memory T cells that are specific for Leptospira or Leptospira protein antigens. METHODS. Peripheral blood mononuclear cells (PBMCs) were obtained from patients who had recovered from leptospirosis, as well as from control individuals. PBMCs were assessed for in vitro proliferation, phenotyping, and cytokine production after stimulation with different strains of Leptospira, recombinant LipL32, or overlapping synthetic peptides of different outer membrane proteins. RESULTS. PBMCs from both control subjects and patients produced significant proliferative responses to all Leptospira strains. Proliferation from control PBMCs was significantly greater than responses produced by patient PBMCs. Select strains of Leptospira expanded both T cell receptor (TCR) alphabeta and TCRgammadelta T cells in both control subject and patient PBMCs. Patient and control subject PBMCs produced equivalent levels of tumor necrosis factor alpha and interferon gamma, but patient PBMCs produced significantly less interleukin 10 than did control subject PBMCs after stimulation by different strains of Leptospira. PBMCs from patients failed to respond to recombinant LipL32 or to any of the Leptospira peptides. CONCLUSION. Leptospira induced significant proliferative responses, TCRgammadelta T cell expansion, and cytokine production in both control subject and patient PBMCs. Patient PBMCs failed to recognize Leptospira protein antigens. Leptospirosis does not seem to generate memory T cells that can be activated by in vitro stimulation.

Figure 1

Live Leptospira- induced proliferation of control PBMC. PBMC were cultured in microtiter wells at 2×10⁵/well with one of the following: 1) medium only, 2) PPD (5µg/mL), or 3) Leptospira, or 4) formalin- fixed Leptospira. Cell proliferation was assessed at 6 days by measuring the amount of [³H] thymidine incorporation. Results are from one experiment that was representative of 7 independent experiments (7 different controls). * P<0.05 live vs fixed bacteria.

Figure 2

Different Leptospira strains induce proliferation of control PBMC. PBMC were cultured in microtiter wells at 2×10⁵/well with one of the following: 1) medium only, 2) PPD (5µg/mL) or 3) different strains of Leptospira (1×10⁷). Cell proliferation was assessed at 6 days by measuring the amount of [³H] thymidine incorporation. Results are from one experiment that was representative of 6 independent experiments (6 different controls).

Figure 3

Different Leptospira strains induced proliferation of control versus patients PBMC. PBMC were cultured in microtiter wells at 2×10⁵/well with one of the following: 1) medium only, 2) PPD (5µg/mL) or 3) Var10, RGA, or HAI188 (1×10⁷). Cell proliferation was assessed at 6 days by measuring the amount of [³H] thymidine incorporation. Results are mean of 13 experiments (13 different patients and 13 different controls) . * P<0.05 Patients vs Controls.

Figure 4

Preferential expansion of TCRαβ⁺ (A) and TCRγδ⁺ (B) T cells. Patient and control PBMC were set up in 24 well culture wells (10⁶/well) and stimulated with Var10 vs RGA vs HAI188 (1×10⁸ ). Cultures were terminated on day 6, and cells obtained from each culture were phenotyped by flow cytometry. Data shown are the mean of 13 experiments (13 different patients and 13 different controls) . * P<0.05 difference between responses of patient PBMC versus control PBMC. Panel A presents TCRαβ T cells Panel B presents TCRγδ T cells.

Figure 5

Cytokine profile produced by PBMC obtained from controls versus patients. PBMC were stimulated with one of the following: 1) medium only, 2) PPD (5µg/mL) or 3) Leptospira (1×10⁸): Var10 vs RGA vs HAI188. Culture supernatants were collected at six days of culture and assessed for different cytokine levels. Panel A: TNF-α, Panel B: IFN-γ and Panel C: IL-10. Data presented are the mean of 13 experiments (13 different patients and 13 different controls) . * P<0.05 difference between patients and controls.

Figure 5

Cytokine profile produced by PBMC obtained from controls versus patients. PBMC were stimulated with one of the following: 1) medium only, 2) PPD (5µg/mL) or 3) Leptospira (1×10⁸): Var10 vs RGA vs HAI188. Culture supernatants were collected at six days of culture and assessed for different cytokine levels. Panel A: TNF-α, Panel B: IFN-γ and Panel C: IL-10. Data presented are the mean of 13 experiments (13 different patients and 13 different controls) . * P<0.05 difference between patients and controls.

Figure 6

PBMC from patients fail to respond to Leptospira antigens. PBMC from leptospirosis recovering patients and control subjects were cultured with one of the following: 1) medium only, 2) PPD, 3) RGA (1×10⁷), 4) LipL32 recombinant protein, or 5) overlapping peptides to LipL32, LipL21, OmpL1, and LipL41. Data presented is the Proliferation Index at day 6 of culture. Panel A: LipL32 recombinant and synthetic peptides, Panel B: LipL21, OmpL1 and LipL41 synthetic peptides. Peptides and recombinant LipL32 were used a 10 µg/ml. Data presented are from one experiment that was representative of 10 different experiments (10 different patients and 10 different controls). P=pool *P<0.05 Patients vs Control PBMC.

Figure 6

PBMC from patients fail to respond to Leptospira antigens. PBMC from leptospirosis recovering patients and control subjects were cultured with one of the following: 1) medium only, 2) PPD, 3) RGA (1×10⁷), 4) LipL32 recombinant protein, or 5) overlapping peptides to LipL32, LipL21, OmpL1, and LipL41. Data presented is the Proliferation Index at day 6 of culture. Panel A: LipL32 recombinant and synthetic peptides, Panel B: LipL21, OmpL1 and LipL41 synthetic peptides. Peptides and recombinant LipL32 were used a 10 µg/ml. Data presented are from one experiment that was representative of 10 different experiments (10 different patients and 10 different controls). P=pool *P<0.05 Patients vs Control PBMC.