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

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

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 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

Receiver operating characteristic (ROC) curves for the ML-CD64 index and the Monocyte-CD64 index.

<p>A) The solid line shows the result for the ML-CD64 index value comparing patients with active TB disease to HD. B) The solid line shows the result for the ML-CD64 index value comparing patients with active TB disease to LTBI subjects. C) The solid line shows the result for the monocyte-CD64 index value comparing patients with active TB disease to HD. D) The solid line shows the result for themonocyte-CD64 index value comparing patients with active TB disease to LTBI subjects.</p

Surface molecules expression on circulating monocytes of patients with active TB disease, LTBI subjects, cured TB patients and HD.

<p>A) Cumulative data of the percentage expressed as median of surface expression and IQR. B) Geometric mean fluorescence intensity (geo-mean) of different surface molecules on monocytes and S.E. C) Representative FACS analysis of surface markers expression on circulating monocytes of one representative subject from each cohort group. D) Receiver operating characteristic (ROC) curve for the MFI value of CD64. The solid line shows the result for the value of comparing active TB <i>vs</i> LTBI.</p

M/L ratio, monocyte and lymphocyte absolute counts of the subjects enrolled.

<p>M/L ratio, monocyte and lymphocyte absolute counts of the subjects enrolled.</p

Correlation between the ML ratio and absolute monocyte and lymphocyte counts.

<p>A) The potential association between the ML ratio and absolute monocyte count, were analyzed by Spearman rank correlation test. Data shown are the values of the individual subjects. B) The potential association between the ML ratio and absolute lymphocyte count were analyzed by Spearman rank correlation test. Data shown are the values of the individual subjects. C) Receiver operating characteristic (ROC) curve for the ML ratio. The solid line shows the result for the value of ML ratio comparing active TB <i>vs</i> HD. D) The solid line shows the result for the value of ML ratio comparing active TB patients with LTBI subjects.</p

Receiver operating characteristic (ROC) curve for the monocyte-CD64 index.

<p>The solid line shows the result for the monocyte-CD64 value comparing patients with active TB disease to cured TB patients.</p