Rapid culling of the CD4+ T cell repertoire in the transition from effector to memory

Abstract

Requirements for CD4+ T cell memory differentiation were analyzed with adoptively transferred SMARTA T cell receptor (TCR) transgenic cells specific for alymphocytic choriomeningitis virus (LCMV) epitope. LCMV-induced effector and memory differentiation of SMARTA cells mimicked the endogenous CD4+ T cell response. In contrast, infection with a recombinant Listeria expressing the LCMV epitope, although resulting initially in massive SMARTA expansion, led to loss of effector function and rapid cell death characterized by high expression of the apoptosis regulator Bim. Defective memory differentiation was seen after stimulation of naive but not memory SMARTA cells, was independent of precursor frequency, and correlated with a lower TCR avidity compared to endogenous responders. In addition, long-lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4+ T cell memory differentiation and longevity depend on the strength of the TCR signal during the primary response.

Figure 1

Adoptively transferred SMARTA cells mimic the endogenous CD4 response to LCMV. a) 1 × 10⁴ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺). Mice were infected with LCMV one day later, and the subsequent expansion, contraction and memory maintenance of SMARTA cells in the spleen were calculated based on expression of Thy1.1. Error bars display the SEM (n=3-4 per time point) and results are representative of four different experiments. b) Splenocytes harvested at day 8 or day 300 post-infection were restimulated with GP_61-80 peptide for four hours in the presence of Brefeldin A. Cells were then stained for intracellular expression of IFNγ, TNFα and IL-2. We gated on CD4⁺ Thy1.1⁺ SMARTA responders and CD4⁺Thy1.1^- endogenous responders from the same animal and assessed their ability to make each cytokine at each time point. Flow plots are representative of effector and memory time points over the course of four time-course experiments.

Figure 2

SMARTA cells expand but fail to gain full effector function and form memory after Lm-gp61 infection. a) 1 × 10⁴ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺). Mice were infected with Lm-gp61 or LCMV one day later, and the expansion, contraction and memory maintenance of SMARTA cells in the spleen were assessed. Representative flow plots display the frequency of Thy1.1⁺CD4⁺ SMARTA cells in the spleen at day 8 and day 60 post-infection for each infection. b) Representative flow plots display the frequency of IFNγ-producing endogenous CD4+ responders (CD4⁺Thy1.1^-) in the spleen following peptide restimulation in the presence of Brefeldin A. c) Representative flow plots compare the ability of SMARTA cells (CD4⁺Thy1.1⁺) and endogenous CD4⁺ responders (CD4⁺Thy1.1^-) in the spleen to make IFNγ, TNFα and IL-2 following peptide restimulation at day 8 post-infection. Flow plots are representative of six separate experiments. d) The number of SMARTA (Thy1.1⁺) and endogenous (IFNγ-producing) responders in the spleen are shown for day 8 and day 60. Error bars are the SEM (n=3/group).

Figure 3

Following Lm-gp61 infection, naïve SMARTA cells lose function and disappear while memory SMARTA differentiate normally. a) 1 × 10⁴ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺). Mice were infected with Lm-gp61 one day later, and SMARTA cells were subsequently tracked in the spleen at several time points post infection based on Thy1.1 expression (left column). Cytokine production by SMARTA (middle column) and endogenous (right column) CD4 responders from the same animal was assessed at the indicated points. Flow plots are representative of three mice per time point in this experiment, and similar results were found in three separate experiments. b) 1 × 10⁴ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺). Mice were infected with LCMV one day later and then rechallenged with Lm-gp61 180 days after the primary infection. Representative flow plots display the frequency of SMARTA cells in the spleen at 7 and 42 days post-rechallenge (top row). The bottom row displays the ability of SMARTA cells at each time point to produce IFNγ and TNFα in response to ex vivo peptide restimulation. Plots are representative of 3-4 mice per group.

Figure 4

SMARTA cells fail to differentiate into memory following Lm-gp61 infection regardless of naïve precursor frequency. a) 1 × 10³, 1 × 10⁴ or 1 × 10⁵ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺) that were infected with Lm-gp61 one day later. Representative flow plots display the frequency of Thy1.1⁺ SMARTA cells among total CD4+ T cells at days 7 and 50 post-infection for each cell dose. b) Bar graph displays the number of SMARTA cells harvested at day 7 post-infection following transfer of the indicated number of naïve SMARTA cells. c) Bar graph displays the fold expansion of SMARTA cells at each cell dose during the first week post-infection. Starting numbers were calculated based on a 10% “take” of transferred cells. Error bars are the SEM (n=3/group).

Figure 5

SMARTA responders demonstrate lower functional avidity, as compared to endogenous CD4 responders to Lm-gp61 in the same host. a) B6 mice were infected with either LCMV or Lm-gp61. Eight days later splenocytes were restimulated over a range of GP_61-80 peptide concentrations and stained for expression of IFNγ and TNFα. Representative flow plots are gated on CD4⁺ T cells and show the frequency of cytokine producers following restimulation at the indicate peptide concentrations. b) The graph displays the number of endogenous CD4⁺ T cells in the spleen capable of producing IFNγ in response to decreasing concentrations of peptide. c) 1 × 10⁴ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺), and mice were infected with LCMV one day later. The graph displays the percent maximal endogenous or SMARTA CD4 response induced by ex vivo peptide restimulation, as measured by the frequency of IFNγ-producing responders at each peptide concentration divided by the frequency of IFNγ-producing responders at the highest peptide concentration (1 × 10^-5 M). d) 1 × 10⁴ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺), and mice were infected with Lm-gp61 one day later. The graph displays the percent maximal endogenous or SMARTA CD4 response induced by ex vivo peptide restimulation, as measured by the frequency of IFNγ-producing responders at each peptide concentration divided by the frequency of IFNγ-producing responders at the highest peptide concentration (1 × 10^-5 M). The error bars are the SEM (n=3 for each group). Results are representative of four separate experiments. e) 1 × 10³ CD44^lo SMARTA cells (Thy1.1⁺) were transferred into B6 hosts (Thy1.2⁺), and mice were infected with LCMV or Lm-gp61 one day later. Decreasing concentrations of gp_66-77/I-A_b Class II tetramer was used to stain SMARTA and endogenous responders at day 8 post-infection. The graph displays the number of tetramer-positive endogenous cells detected at each concentration, normalized to staining with the control tetramer hCLIP/I- A^b. f) and g) The graph displays the percentage of tetramer-positive cells as compared to the number of tetramer-positive cells at the highest tetramer concentration following LCMV or Lm-gp61 infection. Error bars are the SEM (n=4/group).

Figure 6

CD4⁺ memory T cells skew to higher functional avidity over time. a) B6 mice were infected with LCMV. At 8 or 30 days post-infection, splenocytes were restimulated over a range of GP_61-80 peptide concentrations ex vivo and stained for expression of IFNγ and TNFα. The graph displays the percent maximal endogenous CD4 response induced by ex vivo peptide restimulation, as measured by the frequency of cytokine-producing responders at each peptide concentration divided by the frequency of cytokine-producing responders at the highest peptide concentration (1 × 10^-5 M). b) Similar experiments were performed following Lm-gp61 infection. c) Similar experiments were performed as in a, except that 1 × 10⁴ CD44^lo SMARTA cells were transferred one day prior to LCMV infection and the functional avidity of Thy1.1⁺ SMARTA cells was assessed. d) and e) Experiments similar to those in a and b were performed, except that functional avidity was assessed at days 60 and 180 post-infection.