Effector-like CD8⁺ T cells in the memory population mediate potent protective immunity

Abstract

The CD8⁺ memory T cell population is heterogeneous, and it is unclear which subset(s) optimally mediate the central goal of the immune system-protection against infection. Here we investigate the protective capacities of CD8⁺ T cell subsets present at the memory stage of the immune response. We show that a population of CD8⁺ T cells bearing markers associated with effector cells (KLRG1(hi), CD27(lo), T-bet(hi), Eomes(lo)) persisted to the memory phase and provided optimal control of Listeria monocytogenes and vaccinia virus, despite weak recall proliferative responses. After antigen-specific boosting, this population formed the predominant secondary memory subset and maintained superior pathogen control. The effector-like memory subset displayed a distinct pattern of tissue distribution and localization within the spleen, and their enhanced capacity to eliminate Listeria involved specialized utilization of cytolysis. Together, these data suggest that long-lived effector CD8⁺ T cells are optimal for protective immunity against certain pathogens.

Figure 1. CD27^lo CD43^lo CD8⁺ memory T cells display a unique phenotype

(A) Mice were infected with LCMV. One month post-infection, splenocytes were stained for CD8⁺, gp33-D^b tetramer, and the indicated markers. Data is representative of at least 4 experiments (n=12). (B) shows compiled data from experiments in which mice were infected with LCMV, LM-OVA or LM-B8R. At the indicated time periods, antigen specific cells were identified (using gp33-D^b, Ova-K^b or B8R-K^b tetramers, respectively) and the frequency of indicated subsets was determined. Data is compiled from at least 4 experiments (n=14 for 1–1.5 month period, n=25 for 2–4 month period), presented as mean +/− SEM. (C) gp33-D^b tetramer-binding CD8⁺ T cells from spleen and lymph nodes were stained with the indicated markers one month post-infection with LCMV. Data is representative of 4 experiments (n=12). See also Figure S1.

Figure 2. Robust protection by CD27^lo CD43^lo memory cells

(A) Experimental schematic. CD8⁺ CD44^hi LCMV memory cells were sort purified 1–3 months post-infection according to CD27 and CD43 expression and each population injected into naïve recipients, such that an equal number of gp33-D^b tetramer-binding cells were adoptively transferred. (B and C) Mice were challenged with LM-gp33. Five days after challenge the number of CFU of LM in the spleen and liver was determined (B) and the number of gp33-D^b specific CD8⁺ T cells in the spleen quantified (C). Data are from compiled from 3 independent experiments. Data in (B) shows individual CFU, with mean values indicated by a line, while the data in (C) shows mean +/− SD. P-values are represented as follows: ***, p<0.001; **, p<0.01; *, p<0.05. (D) Mice were primed with ActA LM-OVA, and at least one month later memory subsets were sorted and 3 × 10⁴ cells of each subset adoptively transferred per recipient. Host animals (and “No-transfer” controls) were infected with virulent LM-OVA and protection determined at 3, 5 or 7 days post-infection. Data are compiled from 3 experiments, and shown as mean +/− SEM. Statistical analysis is represented by asterisks, indicating significant changes in comparison with the no-transfer group for each time point. (E) A similar experimental plan to (A) was used, except that mice were infected with LM-B8R to induce a B8R-K^b specific response and, following transfer of sorted memory CD8⁺ T cells recipient mice were challenged with VV-WR. Three days after VV-WR infection, the number of PFU of VV in the ovaries was determined. Individual mice compiled from 3 experiments are shown. See also Figure S2

Figure 3. CD27^lo CD43^lo cells are strongly increased with boosting

(A) Example staining of the frequency of K^b-OVA+ cells expressing CD27 and CD43, 3–4 months post-infection with LM-OVA Att (primary memory) or 3–4 months after boosting with LM-OVA (secondary memory). Data is representative of 3 experiments (n=9). (B) Compiled data of the frequency of CD27^hi CD43^lo and CD27^lo CD43^lo within K^b-OVA+ cells. Bars show the average +/− SD from 3 experiments (n=9). (C) MFI of phenotypic markers on primary and secondary memory populations. Naïve (CD44^lo) CD8⁺ T cells and/or B cells (CD19+) are also included for comparison. Bars show the mean +/− SD from 3 experiments (n=9). See also Figure S3

Figure 4. Boosting increases the number of protective memory cells

CD27^hi CD43^lo and CD27^lo CD43^lo phenotype memory CD8⁺ T cells were sorted from primary and secondary memory mice 2.5 – 3.5 months after the last antigen stimulation. Equal numbers of OVA-K^b tetramer staining cells were transferred into normal recipients, which were challenged with LM-OVA one day later. Five days following LM-OVA challenge, animals were sacrificed and (A) CFU of LM was determined in the spleen. (B) The expansion of donor CD27^hi CD43^lo and CD27^lo CD43^lo secondary memory cells was determined in the spleen 5 days post LM-OVA challenge. The dotted line indicates the limit of detection. Data are shown as mean +/− SD and are compiled from two independent experiments.

Figure 5. CD27^lo CD43^lo memory cells localize to the red pulp and non-lymphoid sites

(A) Discrimination of splenic red and white pulp by injection of anti-CD8⁺α antibody. CD27 and CD43 expression on gp33-D^b tetramer-binding CD8⁺ T cells within each compartment is shown, from mice infected 1–2 months earlier with LCMV. A representative example is displayed. (B) Compiled data showing the localization of CD27^hi CD43^lo and CD27^lo CD43^lo memory CD8⁺ T cells, 1–2 months after LM-OVA or LCMV infection (gating on OVA-K^b and gp33-D^b tetramer binding cells, respectively) (n=13). The data represent mean +/− SEM. (C) The frequency of CD27^hi or CD27^lo antigen-specific memory CD8⁺ T cells in the blood, LN, IEL and LPL compartments was determined, 1–2 months after LCMV or LM-OVA infection. Bars show compiled data from 4 experiments (n=9) and are shown as mean +/− SEM. See also Figure S4.

Figure 6. Cytolytic function is required for enhanced protective immunity by CD27^lo CD43^lo cells

(A) Intracellular staining of granzyme B by indicated memory subsets of gp33-D^b tetramer-binding cells, 30 days post LCMV infection. (B) Granzyme B expression in OVA-K^b tetramer staining memory CD8⁺ T cell subsets isolated from animals immunized 3–4 months earlier with ActA LM-OVA. Naïve (CD44^lo) CD8⁺ T cells are included as a control. Data is shown as mean +/− SD (n=9). (C) Memory subsets were sorted from ActA LM-OVA infected B6 or Perforin-deficient (Prf1^−/−) mice, 1–2 months post infection. Cells were transferred in equal number into B6.SJL recipients, which were then challenged with LM-OVA. Five days after infection, the number of CFU in the spleen was calculated. Symbols represent individual mice compiled from 3 different experiments. See also Figure S5.