Treponema pallidum Periplasmic and Membrane Proteins Are Recognized by Circulating and Skin CD4+ T Cells

Abstract

Background: Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment.

Methods: Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens.

Results: We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood.

Conclusions: T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.

Keywords: CD4+ T cell; interferon-γ; secondary syphilis; syphilis; tissue-resident memory T cell; vaccine.

Figure 1.

Immune-experienced PBMC respond to Treponema pallidum sonicate. A, Net PBMC activation measured by IFN-γ ELISPOT after stimulation with T. pallidum and mock sonicate. Data points are the average of duplicates. White dots represent participants with HIV. Horizontal lines are median SFU/10⁶ PBMC. P values were calculated by Kruskal-Wallis test between groups. B, Comparison of ELISPOT responses between T. pallidum-exposed and naive individuals analyzed with Fisher exact test. C, PBMC from participant 02. Cells in the indicated gate were sorted and bulk-expanded. D, The resultant polyclonal CD4⁺ T-cell line was tested for reactivity to antigen sonicates. Dot plots show gated live CD3⁺CD4⁺CD8⁻ responder cells. Abbreviations: AIM, activation-induced marker; HIV, human immunodeficiency virus; IFN-γ, interferon-γ; IL-2, interleukin 2; PBMC, peripheral blood mononuclear cell; SFU, spot forming unit; ICS, intracellular cytokine staining.

Figure 2.

Identification of Treponema pallidum CD4⁺ T-cell antigens. TCL were enriched and expanded from blood (A and B) or cultured directly from skin (C) on day 0, day 30, and day 180 after treatment. A, TCL proliferation in response to defined T. pallidum antigens is reported as CPM from participant 17 with early latent syphilis. Controls at right include T. pallidum sonicate (T. pallidum), mock sonicate (mock), and PHA. B and C, Serial T-cell responses to T. pallidum proteins in blood and skin from participant 14 with secondary syphilis. Data are reported as MAD above the median proliferative response to unrelated recombinant proteins. Technical duplicates are shown as black dots and bars are means. T. pallidum antigens with positive responses (2 replicates above calculated threshold in 2 independent assays) are labeled. Abbreviations: CPM, counts per minute; MAD, median absolute deviation; PHA, phytohemagglutinin; TCL, T-cell line.

Figure 3.

Summary of CD4⁺ T-cell antigen discovery. A, Treponema pallidum proteins driving T-cell responses. Participants 14 and 17 include PBMC- and skin-derived TCL from 3 time points. Participant 17 includes subsequent reinfection time point PBMC-derived TCL. B, Predicted localization of T. pallidum CD4⁺ T-cell antigens in the bacterial cell. Abbreviations: PBMC, peripheral blood mononuclear cell; TCL, T-cell line.

Figure 4.

CD4⁺ T-cell epitope mapping and determination of HLA restriction. Representative data from participant 02. A, PBMC-derived TCL reactivity to Tp0684 (MglB-2) peptides at 1 μg/mL. Triplicate data for the active peptide and representative nonreactive peptides are shown. B, Reactivity of polyclonal TCL to titrated peptide (Tp0684 aa 233–245) is blocked by HLA class II DR mAb. C, IFN-γ secretion by the same effector cells in response to 1 μg/mL peptide Tp0684 aa 233–245 using aAPC expressing participant-relevant HLA DR alleles and the control parent cell line, DAP3. D, Summary of T-cell epitopes and available HLA restriction data. Abbreviations: aa, amino acid; aAPC, artificial antigen presenting cell; DMSO, dimethylsulfoxide; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; mAb, monoclonal antibody; OD, optical density; PBMC, peripheral blood mononuclear cell; TCL, T-cell line.