Bronchoalveolar CD4+ T cell responses to respiratory antigens are impaired in HIV-infected adults

Abstract

Rationale: HIV-infected adults are at an increased risk of lower respiratory tract infections. HIV infection impairs systemic acquired immunity, but there is limited information in humans on HIV-related cell-mediated immune defects in the lung.

Objective: To investigate antigen-specific CD4(+) T cell responses to influenza virus, Streptococcus pneumoniae and Mycobacterium tuberculosis antigens in bronchoalveolar lavage (BAL) and peripheral blood between HIV-infected individuals and HIV-uninfected Malawian adults.

Methods: We obtained BAL fluid and blood from HIV-infected individuals (n=21) and HIV-uninfected adults (n=24). We determined the proportion of T cell subsets including naive, memory and regulatory T cells using flow cytometry, and used intracellular cytokine staining to identify CD4(+) T cells recognising influenza virus-, S pneumoniae- and M tuberculosis-antigens.

Main results: CD4(+) T cells in BAL were predominantly of effector memory phenotype compared to blood, irrespective of HIV status (p<0.001). There was immune compartmentalisation with a higher frequency of antigen-specific CD4(+) T cells against influenza virus, S pneumoniae and M tuberculosis retained in BAL compared to blood in HIV-uninfected adults (p<0.001 in each case). Influenza virus- and M tuberculosis-specific CD4(+) T cell responses in BAL were impaired in HIV-infected individuals: proportions of total antigen-specific CD4(+) T cells and of polyfunctional IFN-γ and TNF-α-secreting cells were lower in HIV-infected individuals than in HIV-uninfected adults (p<0.05 in each case).

Conclusions: BAL antigen-specific CD4(+) T cell responses against important viral and bacterial respiratory pathogens are impaired in HIV-infected adults. This might contribute to the susceptibility of HIV-infected adults to lower respiratory tract infections such as pneumonia and tuberculosis.

Figure 1

Lower proportion of CD4⁺ T cells in BAL and peripheral blood of HIV-infected individuals compared to HIV-uninfected adults. T lymphocytes obtained from BAL and peripheral blood were stained with anti-CD3 FITC, anti-CD4 Pacific blue and anti-CD8 APC-H7 fluorochrome conjugated antibodies. (A) The data shows a lower proportion of CD4⁺ T cells in BAL and peripheral blood of HIV-infected individuals compared to HIV-uninfected adults. (B) The data shows a higher proportion of CD8⁺ T cells in BAL and peripheral blood of HIV-infected individuals compared to HIV-uninfected adults. Significance was assessed using Mann–Whitney U test. Black horizontal bars represent median and IQRs.

Figure 2

The proportions of naive and memory T cell subsets are different between BAL and peripheral blood, and are altered during HIV infection. T lymphocytes obtained from BAL and peripheral blood were stained with anti-CD3 FITC, anti-CD4 Pacific blue, anti-CD8 APC-H7, anti-CD45RA PE and anti-CCR7 APC fluorochrome conjugated antibodies. The proportion of naïve (CD45RA⁺CCR7⁺), central memory (CD45RA⁻CCR7⁺), effector memory (CD45RA⁻CCR7⁻) and terminal effector (CD45RA⁺CCR7⁻) were defined. (A, B, C, D) The data shows that BAL T cells (upper) were predominantly of effector memory phenotype compared to peripheral blood (lower), in which T cells were distributed among naive, central memory, effector memory and terminal effector phenotypes. (A, B) The data shows a higher proportion of effector memory and lower proportion of central memory BAL CD4⁺ (left) and CD8⁺ (right) T cells in HIV-infected individuals compared to HIV-uninfected adults. (C, D) The data shows no difference in peripheral blood CD4⁺ T cell subsets between HIV-infected individuals compared to HIV-uninfected adults (left), but there was a higher proportion of CD8⁺ effector memory and a lower proportion of CD8⁺ terminal effector in HIV-infected individuals compared to HIV-uninfected adults (right). Black horizontal bars represent median and IQRs. Statistical significance was analysed by the Mann–Whitney U test. p value <0.05 was used to determine statistical significance.

Figure 3

Higher frequency of regulatory T cells in BAL compared to peripheral blood, but altered in HIV-infected individuals. T lymphocytes obtained from BAL and peripheral blood were stained with anti-CD4 Pacific blue, anti-CD25 FITC and anti-Foxp3 PE fluorochrome conjugated antibodies. Regulatory T cells (Tregs) were defined as CD4⁺ T cells expressing CD25^hi and FoxP3⁺. (A) A flow cytometry representative dot plot showing Tregs in BAL and peripheral blood from a healthy control. (B) The data shows a higher frequency of Tregs in BAL than peripheral blood in HIV-uninfected adults. It also shows a higher frequency of peripheral blood Tregs in HIV-infected individuals compared to HIV-uninfected adults. (C) The data shows no difference in the absolute counts of Tregs in peripheral blood between HIV-infected individuals and HIV-uninfected adults. Black horizontal bars represent median and IQRs. Statistical significance was analysed by the Mann–Whitney U test in the HIV-uninfected versus HIV-infected comparison, and Wilcoxon Signed rank test in the BAL versus peripheral blood comparison. p value <0.05 was used to determine statistical significance.

Figure 4

Representative flow cytometry dot flow from an HIV-uninfected adult showing multiple subsets of antigen-specific CD4⁺ T cells in BAL and peripheral blood. BAL and peripheral blood lymphocytes were stimulated with antigens and T cell responses were measured by intracellular cytokine staining. Representative flow cytometry dot plots from an HIV-uninfected adult showing interferon-γ (IFN-γ) and TNF-alpha (TNF-α) responses in BAL (top) and peripheral blood (bottom) cells, in an unstimulated negative control and cells stimulated with influenza virus, S. pneumoniae and M tuberculosis antigens.

Figure 5

Lower frequency of antigen-specific BAL CD4⁺ T cells in HIV-infected individuals compared to HIV-uninfected adults. BAL and peripheral blood lymphocytes were stimulated with antigens and the magnitude of the antigen-specific T cell response was measured by intracellular cytokine staining. The total of all cytokine-secreting CD4⁺ T cells was used to represent the percentage frequency of antigen-specific cells. (A) The data shows a higher percentage frequency of influenza virus antigen-specific CD4⁺ T cells in BAL compared to peripheral blood in HIV-uninfected adults. It also shows a lower percentage frequency of BAL influenza virus antigen-specific CD4⁺ T cells in HIV-infected individuals compared to HIV-uninfected adults. (B) The data shows a higher percentage frequency of S pneumoniae antigen-specific CD4⁺ T cells in BAL compared to peripheral blood in HIV-uninfected adults. It also shows a higher percentage frequency of S pneumoniae antigen-specific peripheral blood CD4⁺ T cells in HIV-infected individuals compared to HIV-uninfected adults. (C) The data shows a higher percentage frequency of M tuberculosis antigen-specific CD4⁺ T cells in BAL compared to peripheral blood in HIV-uninfected adults. It also shows a lower percentage frequency of BAL M tuberculosis antigen-specific CD4⁺ T cells in HIV-infected individuals compared to HIV-uninfected adults. Black horizontal bars represent median and IQRs after background responses were subtracted from all the antigen-specific CD4 T cell responses. Statistical significance was analysed by the Mann–Whitney U test in the HIV-uninfected versus HIV-infected comparison, and Wilcoxon Signed rank test in the BAL versus peripheral blood comparison. p value <0.05 was used to determine statistical significance.

Figure 6

Lower proportion of polyfunctional antigen-specific CD4⁺ T cells in BAL and peripheral blood of HIV-infected individuals compared to HIV-uninfected adults. BAL and peripheral blood lymphocytes were stimulated with antigens and the quality of the antigen-specific T cell response was measured by intracellular cytokine staining. The proportion of single producers (IFN-γ alone or TNF-α alone) and double producers (IFN-γ and TNF-α) in the antigen-specific CD4⁺ T cell population was defined. (A) The data shows that in both BAL (upper) and peripheral blood (lower), the proportion of double producers (green) in the influenza virus antigen-specific CD4⁺ T cell population was lower in HIV-infected individuals (right) than HIV-uninfected adults (left). It also shows that the proportion of subsets of antigen-specific CD4⁺ T cells against influenza virus including IFN-γ single producers (blue), TNF-α single producers (red), and IFN-γ/TNF-α double producers (green) were different between BAL (upper) and peripheral blood (lower) in HIV-uninfected adults. (B) The data shows that the proportion of subsets of antigen-specific CD4⁺ T cells against S pneumoniae (including single producers and double producers), were different between BAL (upper) and peripheral blood (lower) in HIV-uninfected adults. (C) The data shows that in BAL (upper) and peripheral blood (lower) the proportion of double producers (green) in the M tuberculosis antigen-specific CD4 T cell population was lower in HIV-infected individuals (right) than HIV-uninfected adults (left). It also shows that the proportion of subsets of antigen-specific CD4⁺ T cells against M tuberculosis (including single producers, and double producers), were different between BAL (upper) and peripheral blood (lower) in HIV-uninfected adults.