Abstract
Within overall Th1-like human memory T cell responses, individual T cells may express only some of the characteristic Th1 cytokines when reactivated. In the Th1-oriented memory response to influenza, we have tested the contributions of two potential mechanisms for this diversity: variable expression of cytokines by a uniform population during activation, or different stable subsets that consistently expressed subsets of the Th1 cytokine pattern. To test for short-term variability, in vitro-stimulated influenza-specific human memory CD4+ T cells were sorted according to IL-2 and IFNγ expression, cultured briefly in vitro, and cytokine patterns measured after restimulation. Cells that were initially IFNγ+ and either IL-2+ or IL-2- converged rapidly, containing similar proportions of IL-2-IFNγ+ and IL-2+IFNγ+ cells after culture and restimulation. Both phenotypes expressed Tbet, and similar patterns of mRNA. Thus variability of IL-2 expression in IFNγ+ cells appeared to be regulated more by short-term variability than by stable differentiated subsets. In contrast, heterogeneous expression of IFNγ in IL-2+ influenza-specific T cells appeared to be due partly to stable T cell subsets. After sorting, culture and restimulation, influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ cells maintained significantly biased ratios of IFNγ+ and IFNγ- cells. IL-2+IFNγ- cells included both Tbetlo and Tbethi cells, and showed more mRNA expression differences with either of the IFNγ+ populations. To test whether IL-2+IFNγ-Tbetlo cells were Thpp cells (primed but uncommitted memory cells, predominant in responses to protein vaccines), influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ T cells were sorted and cultured in Th1- or Th2-generating conditions. Both cell types yielded IFNγ-secreting cells in Th1 conditions, but only IL-2+IFNγ- cells were able to differentiate into IL-4-producing cells. Thus expression of IL-2 in the anti-influenza response may be regulated mainly by short term variability, whereas different T cell subsets, Th1 and Thpp, may contribute to variability in IFNγ expression.
Highlights
The major immune mechanism of protective immunity to influenza virus is often considered to be neutralizing antibody, partly because the seasonal variation in influenza strains involves mainly antibody epitopes
Stable and rapidly-randomizing cytokine patterns in human memory Th1-like cells To test the variability of cytokine patterns in human responses, we measured the ex vivo cytokine patterns expressed by CD4 T cells stimulated by the polyclonal activator SEB
Cell populations initially expressing 2+c+ or 2-c+ patterns, contained similar proportions of IL-2+ and IL-2- cells after re-stimulation (Fig 1A, top two panels). This rapid convergence of the distinct sorted phenotypes into mixed IL-2-/+ populations was consistent with stochastic expression of IL-2 by the IFNc+ cells. This instability of IL-2 expression was observed in additional subjects, with no significant difference between the levels of IL-2 produced on re-stimulation of the IL-2+ versus IL-2- sorted populations (Figure 1B)
Summary
The major immune mechanism of protective immunity to influenza virus is often considered to be neutralizing antibody, partly because the seasonal variation in influenza strains involves mainly antibody epitopes. CD4 cells could provide help to induce antibody production by B cells, induce effector CD8 T cells, and protect the host by helper-independent mechanisms [4,6]. T cell responses may be significant as a cause of immunopathogenesis [4,7]. Influenza-specific human CD4 T cell memory responses are biased strongly towards Th1 cells, producing TNFa, IFNc and IL-2 on activation. IFNc contributes indirectly and directly to protection, inhibits the Th17 response and can cause immunopathogenesis [4]. Not all influenza-specific CD4 T cells express both IL-2 and IFNc
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