Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection

Abstract

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H(+) NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H(+) NK cells, may be not be as robust as the secondary response observed in T cells.

FIGURE 1

Kinetic profiles of endogenous CD4⁺ T cells, endogenous CD8⁺ T cells, endogenous NK cells, and adoptively transferred NK cells. For the endogenous response, C57BL/6 mice were infected with MCMV and CD4⁺ T cell (A), CD8⁺ T cell (B), or NK cell (C) numbers tracked at various time points postinfection. MCMV specificity was determined via IFN-γ staining after stimulation with MCMV lysate (CD4⁺ T cells, filled upright triangles) or peptides (CD4⁺ and CD8⁺ T cells; the stimulating peptides are indicated). The endogenous NK cells responding to MCMV are tracked with KLRG1⁺ expression. D, For NK adoptive transfer studies, LY49H⁺ CD45.1 NK cells were transferred into C57BL/6 DAP12-deficient CD45.2 congenic mice, after which recipient mice were infected. The transferred NK cell population was tracked on various days postinfection.

FIGURE 2

Growth and decay rates for NK, CD8, and CD4 T cells. A, The fastest two-point growth rates for CD4 T cells, CD8 T cells, and NK cells in the lung, liver, and spleen for various MCMVAgs. The fastest two-point growth rate is the fastest growth rate observed between adjacent time points during population expansion. NK growth rates are calculated for an endogenous infection and for the adoptive transfer models of 10⁴ and 10⁵ cells per mouse. B, The fastest two-point decay rates for CD4 T cells, CD8 T cells, and NK cells in the lung, liver, and spleen for various MCMVAgs. The fastest two-point decay rate is the fastest decay rate observed between adjacent time points during population contraction.

FIGURE 3

Decay profiles. Three possible models of decay and describe population contraction. Model 1, The population contraction to zero levels with constant t_1/2. As the population will approach zero levels, this decay model is not indicative of stable memory formation. Model 2, The population contracts to nonzero levels with constant t_1/2. If the plateau is higher than background levels, this indicates a stable memory population has formed. Model 3, Biphasic decay in which the total population consists of two subpopulations, one with a fast and one with a slow t_1/2. This decay is indicative of a fast effector decaying population and a slowly decaying memory population.

FIGURE 4

Representative decay model fits to data. We fit all of the three possible models (Fig. 3) of population decay to the experimental data after the peak of the response to determine which of these models best describes the data. More complex models must fit significantly better to justify the additional parameters to be considered the best fit. A representative plot of the fits for the lung, liver, and spleen (columns 1, 2, and 3, respectively), for CD4⁺ T cells, CD8⁺ T cells, endogenous NK cells, and adoptively transferred NK cells (rows 1, 2, 3, and 4, respectively) are shown. The title above each plot describes the model of best fit and/or the representative epitope shown. CD4⁺ T cells, CD8⁺ T cells, and endogenous NK cells fit well to both the exponential with plateau model and the biexponential model (models 2 and 3, respectively, from Fig. 3). Importantly, the endogenous NK cell population has returned to preinfection levels 20 d after the peak. The adoptively transferred NK cell population fits best to a simple exponential model (model 1 from Fig. 3).

FIGURE 5

T cell and NK cell immune response kinetics. A, From a very low precursor frequency, T cells quickly expand to their population peak, after which the population begins to contract. T cell contraction is multiphasic, with a fast initial decay and either a slow late decay or plateau. The T cell memory population level is high compared with the naive precursor frequency, providing long-term protection. B, From a high precursor frequency (~40% of all NK cells), responding Ly49H⁺ NK cells slowly grow to their population peak, which occurs at around the same time as the peak in T cells. After this peak, the responding NK cell population experiences constant, continuous decay and quickly returns to below basal levels. As these Ag-experienced or memory NK cells continue to decay, they will eventually comprise an insignificant proportion of the basal NK levels. Thus, memory NK cells may only provide increased protection for the short term when compared with T cells.