Low expression of activation marker CD69 and chemokine receptors CCR5 and CXCR3 on memory T cells after 2009 H1N1 influenza A antigen stimulation in vitro following H1N1 vaccination of HIV-infected individuals

Abstract

Unlike well-studied antibody responses to pandemic 2009 H1N1 influenza A virus vaccines in human immunodeficiency virus-infected (HIV+) individuals, less well understood are cell-mediated immune (CMI) responses to this antigen in this susceptible population. We investigated such influenza-specific CMI responses in 61 HIV+ individuals and in 20 HIV-negative (HIV-) healthy controls. Each was vaccinated with a single licensed dose of inactivated, split-virion vaccine comprised of the influenza A/California/7/2009 (H1N1) virus-like strain. Cells collected just prior to vaccination and at 1 and 3 months afterwards were stimulated in vitro with dialyzed vaccine antigen and assayed by flow cytometry for cytokines TNF-α, IFN-γ, IL-2, and IL-10, for degranulation marker CD107a, as well as phenotypes of memory T-cell subpopulations. Comparable increases of cytokine-producing and CD107a-expressing T cells were observed in both HIV+ subjects and healthy HIV-controls. However, by 3 months post-vaccination, in vitro antigen stimulation of peripheral blood mononuclear cells induced greater expansion in controls of both CD4 and CD8 central memory and effector memory T cells, as well as higher expression of the activation marker CD69 and chemokine receptors CCR5 and CXCR3 than in HIV+ subjects. We concluded CD4+ and CD8+ memory T cells produce cytokines at comparable levels in both groups, whereas the expression after in vitro stimulation of molecules critical for cell migration to infection sites are lower in the HIV+ than in comparable controls. Further immunization strategies against influenza are needed to improve the CMI responses in people living with HIV.

Keywords: 2009 H1N1 influenza A vaccine; HIV-infection; activation markers; cellular immunity; chemokine receptors; immunological memory responses.

Figure 1.

Flow cytometry of cytokine-producing and CD107a-expressing T cells. (A) CD3+ cell populations identified from CD3-versus-side scatter (SSC) cytometric density plot. (B) Single-positive CD4+ and CD8+ cell populations identified from CD4-vs.-CD8 plot. (C) Plots of IFN-y, IL-2, and IL-10 cytokine production, and surface CD107a expression, versus TNF-a+, expressed in mean fluorescent intensity. Fold increases of TNF-a+ IFN-y, IL-2, and IL-10 cytokine production, and CD107a–expression, by CD4+ (D) and CD8+ (E) in response to vaccine antigen, and by phytohemagglutin (PHA) plus CD4+ as control (F), by HIV-infected (HIV+) and healthy (HIV-) individuals, compared before in vivo vaccination on day zero (D0), and at one month (M1) and 3 months (M3) after vaccination. Medians represented by thick, wide horizontal bars, and 25%–75% interquartile ranges by thin, narrow bars.

Figure 2.

Flow cytometry of cytokine producing memory T cells. (A) T cell populations were identified as CD3+ population from CD3 vs. side scatter (SSC) dot plot. (B) Single positive CD4 or CD8 populations were gated from CD4 versus CD8 dot plot. (C) Cytokine production and expression of surface CD107a of memory (CD45RO+) CD4+ or CD8+ T cell subpopulation were then evaluated. Fold increase of memory CD4+ (D) or CD8+ (E) T cells were compared between HIV-infected (HIV+) and healthy (HIV-) individuals after stimulation at before (D0), one month (M1) and 3 months (M3) after vaccination. Statistical analysis was performed. p-value < 0.05 were considered statistically significant. Solid lines show the medians with interquartile ranges of each group.

Figure 3.

Increases of memory T cell subpopulations in response to pandemic H1N1 influenza A vaccine antigen. (A) T cell populations were identified as CD3+ population from CD3 vs. side scatter (SSC) dot plot. (B) Single positive of CD4 or CD8 population were gated from CD4 versus CD8 dot plot. (C) Central memory (CM; CD45RO+ CD62L+) and effector memory (EM; CD45RO+ CD62L-) T cells were then evaluated from CD45RO vs. CD62L dot plot. The increase of antigen-specific CD4+ T_CM (D) and CD8+ T_CM (E) and CD4+ T_EM (F) and CD8+ T_EM (G) cells were compared between HIV- infected (HIV+) and healthy (HIV-) individuals after stimulation at before (D0), one month (M1) and 3 months (M3) after vaccination. Statistical analysis was performed. p-value < 0.05 were considered statistically significant. *, ** and *** show p < 0.05, 0.01 and 0.001, respectively. Solid lines show the medians with interquartile ranges of each group.

Figure 4.

Expression of activation and inhibition markers of T cells after stimulation with pandemic H1N1 influenza A vaccine antigen. Gating of CD3+ T cells (A) was identified for CD4 or CD8 single positive populations from CD4 versus CD8 dot plot (B). Memory T cells were identified by expression of CD45RO (C) and the expressions of CD28 and CD69 (D) or CTLA4 and PD1 (did not show) were then evaluated. The increase of expressions of CD69 on total CD4+ (E) and CD8+ (F) and memory CD4+ (G) and CD8+ (H) or CTLA4 on total CD4+ (I) and CD8+ (J) T cells were compared between HIV-infected (HIV+) and healthy (HIV-) individuals after stimulation at before (D0), one month (M1) and 3 months (M3) after vaccination. Statistical analysis was performed. p-value <0.05 were considered statistically significant. *, ** and *** show p < 0.05, 0.01 and 0.001, respectively. Solid lines show the medians with interquartile ranges of each group.

Figure 5.

Expression of trafficking molecules on T cells after stimulation with pandemic H1N1 influenza A antigen. Expressions of CCR5, CXCR3 and both molecules on total CD4+ (A, E and I, respectively) or CD8+ (B, F and J, respectively) and effector memory CD4+ (C, G and K, respectively) or memory CD8+ (D, H and L, respectively) T cells were compared between HIV-infected (HIV+) and healthy (HIV-) individuals at before (D0), one month (M1) and 3 months (M3) after vaccination. Statistical analysis was performed. p-value <0.05 were considered statistically significant. *, ** and *** show p < 0.05, 0.01 and 0.001, respectively. Solid lines show the medians with interquartile ranges of each group.