Immune characterization of the HBHA-specific response in Mycobacterium tuberculosis-infected patients with or without HIV infection

<div><p>Introduction</p><p>RD1-based Interferon-γ Release Assays (IGRAs) cannot distinguish latent from active tuberculosis (TB) disease. Conversely, a positive response to heparin-binding haemagglutinin (HBHA)-based IGRAs, among TB-infected subjects, correlates with <i>Mycobacterium tuberculosis</i> (<i>Mtb)</i> containment and low risk of TB progression. The aim of this study was to characterize HBHA-immune responses in HIV-infected and uninfected subjects with active TB or latent TB infection (LTBI).</p><p>Methods</p><p>49 subjects were prospectively enrolled: 22 HIV-uninfected (13 TB, 9 LTBI) and 27 HIV-infected (12 HIV-TB, 15 HIV-LTBI). Whole blood and peripheral blood mononuclear cells were stimulated with HBHA and RD1 antigens. Interferon (IFN)γ release was evaluated by ELISA whereas cytokine profile [IFNγ, tumor necrosis (TNF)α, interleukin (IL)2] and phenotype (CD45RA, CCR7) by flow cytometry.</p><p>Results</p><p>Among LTBI individuals, HBHA stimulation induced IFNγ release in all the HIV-uninfected, while, only 4/15 HIV-infected responded. Within the active TB, only 5/13 HIV-uninfected and 1/12 HIV-TB patients responded. Interestingly, by cytometry we showed that CD4⁺ T-cells response to HBHA was significantly impaired in the HIV-infected subjects with TB or LTBI compared to the HIV-uninfected subjects. The phenotype of HBHA-specific CD4 T-cells showed a predominantly central memory (CM) and effector memory (EM) phenotype without differences among the groups. Differently, HBHA-specific CD8⁺ T-cells, showed mainly a CM and naïve phenotype in LTBI group while TB, HIV-LTBI and HIV-TB groups were characterized by EM or terminally differentiated phenotypes. Interestingly, differently than what observed for RD1, the cytokine profile of HBHA-specific T-cells evaluated by cytometry showed that the CD4⁺ T-cells were mostly monofunctional. Conversely, CD8-specific T-cells were mostly monofunctional for both HBHA and RD1 stimulations.</p><p>Conclusions</p><p>These results characterize the impact of HIV infection in CD4- and CD8-specific response to HBHA in both LTBI and TB patients. HIV infection impairs the CD4 response to HBHA and likely this may lead to an impairment of TB control.</p></div

Analysis of polyfunctional and monofunctional response to different antigens of CD4⁺ and CD8⁺ T-cell subsets in patients enrolled for the study.

<p>The graphs show the frequency of polyfunctional (more than one cytokine) and monofunctional (one cytokine) response, evaluated by flow cytometry, to HBHA (A, B), RD1 proteins (C, D) and CMV (E, F) of CD4⁺ and CD8⁺ T-cell subsets in the <i>Mtb</i>-infected patients with or without HIV infection. A positive cytokine response was defined as at least twice the background. A frequency of any cytokine-producing T-cells (IFNγ and/or TNFα and/or IL2) of at least 0.03% was considered as a positive CD4 and CD8 T-cell response. The horizontal lines represent the median; filled symbols represent polyfunctional T-cells, open symbols represent monofunctional T-cells. Statistical analysis was performed using the Mann-Whitney test and p value was considered significant if < 0.05. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection; HBHA: heparin-binding haemagglutinin; RD: region of difference; CMV: cytomegalovirus.</p

Flow chart of the patients enrolled for the study.

<p>Forty-nine <i>Mtb</i>-infected subjects were prospectively enrolled for the study. Twenty-two patients out of the total were HIV-uninfected, for 13 of these a diagnosis of active TB was made and 9 resulted LTBI. Among the remaining 27 HIV-infected patients, all naïve to ART, 12 were diagnosed as HIV-TB and 15 resulted HIV-LTBI. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection.</p

IFNγ-response to different <i>Mtb</i>-antigens in patients enrolled for the study.

<p>IFNγ-response to different <i>Mtb</i>-antigens was evaluated by short-term stimulation of whole blood in <i>Mtb</i>-infected patients with or without HIV infection. IFNγ from day-1 supernatants were evaluated by commercial ELISA in response to HBHA (A), QFT-IT antigen (B) and Mitogen (C). The square represents LTBI group, the triangle with the bottom tip represents HIV-LTBI group, the dot represents active TB group and the triangle with the top tip represents HIV-TB group. The data are shown after the subtraction of the results obtained in the unstimulated samples. Horizontal lines indicate the median of IFNγ production. Dotted lines indicate the cut-off for either QFT-IT or Mitogen as indicated by the suppliers and for HBHA antigen as for previously shown results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183846#pone.0183846.ref024" target="_blank">24</a>;<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183846#pone.0183846.ref025" target="_blank">25</a>]. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection; HBHA: heparin-binding haemagglutinin; QFT-IT: QuantiFERON-TB Gold (QFT^®) in tube; IFN: interferon; IU: International Unit.</p

IFNγ profile of CD4⁺ and CD8⁺ T-cells in response to different stimuli.

<p>The IFNγ cytokine profile of CD4⁺ and CD8⁺ T-cells was analyzed by ICS. In particular, we evaluated the “total IFNγ response” and then calculated the proportion of this response within the total cytokine response (IFNγ and/or TNFα and/or IL2). PBMC were stimulated overnight with HBHA (A, B), RD1 proteins (C, D), CMV (E, F) and SEB (G, H) and analyzed within the CD4⁺ and CD8⁺ T-cells by flow cytometry. A-H: The bar graphs show the proportion of IFNγ-expressing cells within the CD4⁺ and CD8⁺ T-cells over the total responding T-cells in the different groups analyzed. The horizontal lines represent the median. Statistical analysis was performed using Mann-Whitney test and p value was considered significant if < 0.05. A, C, E, G) Proportion of total IFNγ-producing CD4⁺ T-cells in response to each stimulus; B, D, F, H) Proportion of total IFNγ-producing CD8⁺ T-cells in response to each stimulus. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection; HBHA: heparin-binding haemagglutinin; RD: region of difference; CMV: cytomegalovirus; SEB: staphylococcal enterotoxin B; IFN: interferon.</p

L-arginine metabolism is correlated with the inhibition activity of MDSCs.

<p>Levels of (A) L-arginine, (B) Ornithine and (C) Nitrate in the serum of healthy donors (HD, N.15) and active TB patients (TB, N.30). Results are expressed as the median ± IQR. *P<0.05 **P<0.02.</p

Successful TB treatment reduces the concentration of MDSCs in peripheral blood cells.

<p>(A) Analysis of MDSC frequency in patients with active TB (N.30) and cured TB (N.10). (B) MDSC frequency in cured TB at the end of therapy (N.5) and 1–3 years after the end of therapy (past TB, N.5). Results are expressed as the median ± IQR. *P<0.05.</p

B-cells contribute to the magnitude of T-cell responses in treated TB patients.

<p>To investigate the contribution of B-cells to T-cell activation in the various groups of Mtb infected individuals we depleted CD19⁺ B-cells from PBMCs and compared the T-cell responses following BCG stimulation in total PBMC to those in B-cell depleted PBMCs. Ethnicity of donors was indicated using the following symbols: ‘black circle’ = West Europe; ‘black diamond’ = Est Europe; ‘black square’ = Africa; ‘black triangle’ = Asia; ‘black star’ = Sud America. Data obtained from B-cell depleted samples were compared pair-wise to total PBMC samples using the Wilcoxon signed Rank test and a p-value < 0.05 was considered significant. *: p < 0.05; **: p < 0.01; ***: p < 0.001; @: p = 0.052 (PD1 on CD4⁺ T-cells in LTBI), p = 0.055 (PD1 on CD8⁺ T-cells in TB treated patients s). A. Cytokine production (median in pg/ml) in supernatants on day 5 following BCG stimulation (medium subtracted) for total PBMC (black) and B-cell depleted PBMC (grey). Data are expressed as mean + standard error of the mean. B. Expression of regulatory T-cell markers, or exhaustion markers PD1 and KLRG1 (as percentage of CD4⁺ or CD8⁺ T-cells) in total PBMCs (filled dots) or following CD19 depletion (open dots).</p

B-cells from TB patients as well as LTBI individuals have an impaired function.

<p>Total B-cells were isolated from PBMCs using magnetic bead separation for CD19 and stimulated with the combination of anti-CD40 and anti IgG/ IgM activating antibodies to obtain maximal, polyclonal B-cell activation. Data on isolated B-cells were obtained from 17 controls, 14 LTBI individuals, 13 TB patients and 16 treated TB patients. Lines indicate the median values of all groups. Ethnicity of donors was indicated using the following symbols: ‘black circle’ = West Europe; ‘black diamond’ = Est Europe; ‘black square’ = Africa; ‘black triangle’ = Asia; ‘black star’ = Sud America. Mann-Whitney test was performed to compare infected groups to the uninfected controls and a p< 0.05 was considered significant. * marks differences that remained significant after multiple test correction using Kruskal-Wallis testing with Dunn’s post-test. A. Isolated total CD19⁺ B-cells were labelled with a violet cell proliferation dye, proliferation in unstimulated conditions was subtracted from the anti-CD40 + purified IgG/ IgM stimulated conditions to obtain the delta proliferation reflecting the stimulation induced proliferation. Proliferation was assessed after 6 days of stimulation. B. Cytokine production measured by intracellular cytokine staining by flow cytometry for IL-6 (B) and IL-10 (C). Boxes express the 25–75% of data, with a line at median and whiskers indicate 5–95% data points. Cytokine production was measured after 2 days of stimulation; cytokines in unstimulated conditions were subtracted from stimulated conditions. C. Immunoglobulin production was measured in supernatants of unstimulated, isolated B-cells on day 2 of culture using multiplex bead array and expressed as total pg/ml for all isotypes combined. Lines indicate median values for each group. D. Immunoglobulin production per isotype, expressed as mean production per group of individuals for IgA, IgM, IgE and IgG. E. HLA-DR expression on B-cells following stimulation with anti-CD40 + anti IgG/ IgM expressed within the total CD19⁺ B-cells. Lines indicate median values.</p

Lymphocyte, B cell and monocyte distribution in the lung.

<p>CD14, CD3 and CD20 cell distribution within lung tissues of patients died for TB, pneumonia, or causes other than pneumonia. Representative histological examination of lung specimens from autopsies of control patients died for causes other than pneumonia (n = 5) (a, d, g, j), for bacterial pneumonia (n = 10) (b, e, h, k) or for pulmonary TB (n = 10) (c, f, i, l). Samples were stained with CD14 Ab, CD3 Ab, CD20 Ab and Ki67 Ab. In TB patients B-cells are found mainly around the granuloma (m) or as aggregates near vessels (n). Original magnification (OM), 200x.</p