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. 2008 Oct 17;29(4):650-9.
doi: 10.1016/j.immuni.2008.07.017.

Memory inflation during chronic viral infection is maintained by continuous production of short-lived, functional T cells

Christopher M Snyder  1 , Kathy S Cho, Elizabeth L Bonnett, Serani van Dommelen, Geoffrey R Shellam, Ann B Hill
Affiliations

Memory inflation during chronic viral infection is maintained by continuous production of short-lived, functional T cells

Christopher M Snyder et al. Immunity. .

Abstract

During persistent murine cytomegalovirus (MCMV) infection, the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells, which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation, MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead, we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported, at least in part, by memory T cells primed early in infection, as well as by recruitment of naive T cells at late times. Thus, these data show that memory inflation is maintained by a continuous replacement of short-lived, functional cells during chronic MCMV infection.

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Figures

Figure 1
Figure 1
Inflationary T cells remain functional throughout chronic MCMV infection. A) T cell responses specific for m139, M38 and IE3 inflate over the first 3 months of infection. Shown is the percentage of MCMV-specific T cells with the indicated specificity over time as assessed by MHC-tetramer staining. Each symbol represents an individual mouse. The black line represents the average of all mice in this experiment. Note that the scale is different for M45 and M57-specific T cell populations. B) Similar percentages of antigen-specific T cells are found by both tetramer staining and intracellular cytokine analysis. Peripheral blood from individual mice infected for >3 months was divided and stained with the indicated tetramer or stimulated with the indicated peptide and assessed for IFNγ production. Except for the naïve, tetramer-staining control, the FACS plots shown in each column represent the same mouse. A summary of the data from multiple mice is shown in the graphs to the right. The solid line in each graph represents an ideal 1:1 correlation between tetramer staining and IFNγ production. C) Peripheral blood cells were stimulated as above and stained for IFNγ and TNFα production. D) MCMV-specific T cells can kill targets pulsed with peptides. Naïve CD45.1 congenic mice were stained with varying concentrations of CFSE, loaded with the indicated peptide and transferred i.v. into chronically infected C57BL/6 mice. FACS analysis of the surviving donor cells in the spleen of recipient mice was performed approximately 18 hours later.
Figure 2
Figure 2
Phenotype of stable and inflationary CD8 T cells. Peripheral blood and/or splenocytes from mice infected for >3 months was stained with the indicated tetramer and antibodies specific for CD8 and the indicated cell surface molecule. The plots shown are gated on tetramer+ CD8 T cells (black line) or tetramer negative CD8 T cells from the same sample (shaded histogram). Data is compiled from multiple experiments and multiple time points during chronic infection.
Figure 3
Figure 3
Inflationary T cells fail to divide in SCID recipients, but can respond to a new viral infection. A) Schematic of the adoptive transfer. Splenocytes from C57BL/6 mice infected for >3 months were transferred into C57BL/6-SCID mice or naïve CD45.1 congenic mice. Naïve, CD45.1 congenic recipients were infected with MCMV 2 days later. B) Peripheral blood from SCID recipients (d12 post transfer) or splenocytes from CD45.1 congenic recipients (d7 post infection) were stained with the indicated tetramers. The FACS plots have been gated on CD8+, donor T cells in all cases. C) Cells were FACS sorted for CD8+, CD27 high and low cells. Sorted cells were CFSE labeled and transferred into naïve, congenic mice. Recipients were infected one hour later and donor cells were analyzed in the peripheral blood on day 7 post transfer. Numbers represent the percent of all CD8 T cells that fall into the indicated gates.
Figure 4
Figure 4
Inflationary T cells decline over time in both immune and naïve recipients. A) Splenocytes from C57BL/6 mice infected for >3 months were harvested and transferred into infection matched CD45.1 congenic recipients. Donor, tetramer-positive cells were tracked over time as a percentage of all CD8 cells in the peripheral blood of recipients. B) Extensively proliferated populations of inflationary T cells (CFSE negative based on recipient cells) can be detected in infected recipients, but fail to accumulate over time. Shown are FACS plots at two time points from the same individual mice gated on donor CD8 cells. C) Extensively proliferated populations within the inflated memory T cell pools are only evident in infected animals. Shown is the percentage of extensively divided tetramer positive T cells (CFSE negative as shown in 4B) specific for either stable (left panels) or inflationary (right panels) T cells. The Student's t test was used to assess significance. D) Inflationary T cells decline over time in both infected and naïve recipients. When measured as a percentage of all CD8 T cells, donor CD8 T cells engrafted ~1.5 to 2x better in naïve recipients compared to immune recipients. To correct for these differences in the "take" of the donor population, the percentage of the donor MCMV-specific T cells was normalized to 100% at the first time point. The error bars represent the standard deviation from the average. T cell half-life was estimated by the length of time it took for the population to be reduced by 50% in circulation, relative to all CD8 T cells. E) The total percentage of MCMV-specific T cells is stable after transfer. Shown is the total (donor + recipient) tetramer staining population from the same mice as in D. Error bars represent standard deviation.
Figure 5
Figure 5
Recruitment of naïve T cells occurs during chronic infection. A) C57BL/6 mice that were infected for >3 months were treated with busulfan and injected with bone marrow from naïve CD45.1 congenic donors. Donor, MCMV-specific T cells in the peripheral blood were tracked by intracellular cytokine staining over time. B) Donor-derived MCMV-specific T cells can be found in all recipients. Shown are representative FACS plots gated on all CD8 T cells from mice that received naïve bone marrow 9 weeks earlier. C) Donor MCMV-specific cells represent an increasing percentage of the total peptide-specific response over time. The graphs show donor-derived cells that produced IFNγ in response to the indicated peptides as a percentage of the total peptide-specific IFNγ response. The data was normalized to the amount of donor-derived IFNγ found at the first time point. The lines represent the average of the donor responses over time. Combined data from three independent experiments is shown. D) The percentage of peptide-specific response that is donor derived correlates with the donor CD8 T cell engraftment. Shown is the same data as in C, but graphed with respect to the percent of all CD8 T cells that are donor-derived in the peripheral blood. E) Naïve T cells that develop in the infected host can be recruited into the inflationary pools. The experiment was carried out as in B, but with CD3-depleted donor bone marrow.
Figure 6
Figure 6
Naive T cells do not account for the replacement of all decaying circulating differentiated T cells. A) One mouse in which most of the newly produced T cells were donor derived, but most MCMV-specific T cells were recipient derived 10 months post transplant. B) MCMV-specific chimerism is shown for several mice with a high level of chimerism within double-positive thymocytes C) Donor MCMV-specific cells transferred at the peak of T cell expansion can persist or inflate. Sample FACS plots are from the same mouse at each time point. The numbers indicate the percent of all tetramer+ cells that are donor derived. D) Two examples of dramatic inflation of the donor MCMV-specific T cell populations 7 months after transfer. E) Combined data (as in C and D) from multiple mice and two independent experiments. Shown is the donor tetramer+ population as a percent of all tetramer+ T cells to adjust for inflation of the m139 and M38 populations over time. Lines connect individual mice at either 2 weeks post transfer (3 weeks post infection) or 6–7 months post transfer. IE3-specific T cells were not measured at 2 weeks post transfer.
Figure 7
Figure 7
Model of the dynamics of the inflationary MCMV-specific T cell populations. Ex vivo measurement of MCMV specific T cells at a given time point represents a snap-shot of a highly dynamic population. These T cells undergo a small amount of antigen-driven cell division, but most cells disappear from the blood with a half-life of ~45 to 60 days. The decaying cells are replaced primarily by the progeny of cells that were primed early in infection. We would expect these clones to persist as the individual decaying cells are replaced by the progeny of identical clones that were primed early in infection. However the population is also supported by the recruitment of naïve T cells at late times. As new cells are recruited, we predict that the clonal composition of each inflationary population will change over time. Though the inflationary populations may appear similar at any given time point, our evidence shows that these populations are constantly in flux.
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