Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation

Abstract

In response to infection, CD8(+) T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127(low)KLRG1(high)) and memory precursor effector cells (CD127(high)KLRG1(low)) from an early effector cell that is CD127(low)KLRG1(low) in phenotype. CD8(+) T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12-dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8(+) T cell functionality and memory subset distribution, but in an IL-12-independent manner. Population dynamics were dramatically different during secondary CD8(+) T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127(high)KLRG1(high) memory cells, both of which were intrinsic to the memory CD8(+) T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8(+) T cell differentiation.

Figure 1. Effector cell subset differentiation differs following VSV and LM infection

Mice were infected i.v. with either VSV-ova or LM-ova. At the indicated times the Ova/K^b-specific CD8⁺ T cells in the spleen were monitored for expression of KLRG1 and CD127. (A) Representative plots show the expression of KLRG1 and CD127 on the Ova/K^b-specific CD8⁺ T cells. Plots are representative of 4-5 mice per time-point and three independent experiments. In the upper right corner of each plot is the mean percentage of the population in each quadrant. (B) Graphical depiction of the Ova/K^b-specific effector CD8⁺ T cell sub-population dynamics following LM (left graph) or VSV (right graph) infection. Each dot represents the mean of 4-5 individual mice ± one SEM. Each color represents an effector cell sub-population: CD127^low KLRG1^low, early effector cells (black); CD127^low KLRG1^high, short-lived effector cells (red); CD127^high KLRG1^low, memory-precursor effector cells (blue); CD127^high KLRG1^high (green). These data are representative of 3 independent experiments.

Figure 2. Early effector CD8⁺ T cells have the greatest development potential

Five days after VSV-ova infection CD11a^high CD45.1⁺ OT-I CD8⁺ T cells were sorted into three populations: SLEC (CD127^low KLRG1^high), EEC (CD127^low KLRG1^low), and MPEC (CD127^high KLRG1^high). After which, 10⁴ CD45.1⁺ OT-I CD8⁺ T cells were transferred into infection matched CD45.2⁺ mouse. Either 2 or 35 days later expression of KLRG1 and CD127 was assessed on the transferred CD45.1⁺ OT-I CD8⁺ T cells in the spleen. These data are representative of 2 independent experiments, each containing 3-4 mice per group.

Figure 3. Differential role of IFNAR and IL-12rβin effector CD8⁺ T cell differentiation following VSV and LM infection

(A) Wild-type (CD45.1/2⁺) OT-I CD8⁺ T cells and cytokine receptor deficient (CD45.2⁺) OT-I CD8⁺ T cells were co-transferred to CD45.1⁺ mice. Mice were infected 18 hours later i.v. with either VSV-ova or LM-ova. Seven days later the Ova/K^b-specific CD8⁺ T cells in the PBL were monitored for differentiation of effector CD8⁺ T cell subsets based on KLRG1 and CD127 expression. Three sets of mixed OT-I adoptive transfers were analyzed: WT:IL-12rβ^−/−(B), WT:IFNAR^−/− (C), and WT:IL-12rβ^−/− IFNAR^−/− (D).Each bar represents the median ± one SEM. Each graph represents the mean of 4-5 mice per group and at least 2 independent experiments. Statistical significance was determined using a paired Student’s t-test (*p<0.05; **p<0.01)

Figure 4. ODN1826 enhances SLEC differentiation through an IL-12 and Ebi3 dependent mechanism

(A-C) C57BL/6 and p35^−/− mice were infected i.v. with VSV-ova. After viral infection, some mice were treated with either 50μg ODN1826 (within 2 hours) or 0.5μg rIL-12 (two doses, 1 hour and 24 hours). Seven days later the VSV-N/K^b-specific CD8⁺ T cells in the spleen were identified by tetramer staining. Absolute number of VSV-N/K^b-specific CD8⁺ T cells (A), while effector CD8⁺ T cell differentiation was then assessed based on KLRG1 and CD127 expression by flow cytometry (B) and each effector sub-population was quantified as a percentage of the tetramer⁺ CD8⁺ T cell population (C). (D-E) C57BL/6 and Ebi3^−/− mice were infected i.v. with VSV-ova. After viral infection, some mice were treated with either 50μg ODN1826 (within 2 hours). Seven days later the Ova/K^b-specific CD8⁺ T cells in the spleen were identified by tetramer staining. Absolute number of Ova/K^b-specific CD8⁺ T cells (D) while effector CD8⁺ T cell differentiation was then assessed based on KLRG1 and CD127 expression by flow cytometry (E) and each effector sub-population was quantified as a percentage of the tetramer⁺ CD8⁺ T cell population (F). For panels B &E representative contour plots show the expression of KLRG1 and CD127 on the antigen-specific CD8⁺ T cells. In the upper right corner of each plot is the mean percentage of the population in each quadrant, which is graphed in panels C & F. Statistical significance was determined using a one-way ANOVA (*p<0.05 and **p<0.01). These data are representative of 4-5 mice per group and two independent experiments.

Figure 5. ODN1826 enhances effector CD8⁺ T cell cytokine functionality through an IL-12 independent mechanism

C57BL/6 and p35^−/− mice were infected i.v. with VSV-ova. After viral infection, some mice were treated with either 50μg ODN1826 (within 2 hours) or 0.5μg rIL-12 (two doses, 1 hour and 24 hours). Seven days after infection the potential of CD8⁺ T cells to produce IFNγ and TNFα was assessed by intracellular cytokine staining. Either IFNγ mean fluorescent intensity (A) or the percentage of IFNγ⁺ TNFα⁺ cells (B) was examined. For each panel a representative FACS plot showing the typical cytokine profile and gating is shown. Graphs show the median for 4-5 mice per group and are representative of at least three independent experiments. Statistical significance was determined using a one-way ANOVA and a Newman-Keuls post-test (*p<0.05; **p<0.01; ***p<0.001)

Figure 6. ODN1826 limits CD62L^low MPEC differentiation in an IL-12 independent mechanism

C57BL/6 and p35^−/− mice were infected i.v. with VSV-ova. After viral infection, some mice were treated with either 50μg ODN1826 (within 2 hours) or 0.5μg rIL-12 (two doses, 1 hour and 24 hours). Seven days later the Ova/K^b-specific CD8⁺ T cells in the spleen were identified by tetramer staining. Memoryprecursor CD8⁺ T cells were identified based on KLRG1 and CD127 expression. The proportion of CD62L^highMPECs was assessed (A&B). Additionally, the absolute number of CD62L^high MPEC (C) and CD62L^low MPEC (D) were calculated. Each dot represents an individual mouse from three independent experiments. Statistical significance was determined using a one-way ANOVA and a Newman-Keuls post-test (*p<0.05; **p<0.01; ***p<0.001)

Figure 7. CD8⁺ T cell-intrinsic enhancement of KLRG1 expression during secondary infection

To compare the primary and secondary immune responses, naïve and memory C57BL/6 mice were infected with either VSV-ova or LM-ova. At either the peak of the CD8⁺ T cell response (d5 secondary infection or d7 primary infection) or 2 months post-infection, mice were bled and the Ova/K^b-specific CD8⁺ T cells were identified by tetramer staining and their expression of KLRG1 and CD127 was assessed (A). To determine if the enhanced KLRG1 expression was intrinsic to the memory CD8⁺ T cell, we co-transferred VSV-induced memory OT-I CD8⁺ T cells (CD45.1/2⁺) with naïve OT-I CD8⁺ T cells (CD45.2⁺). One day later those mice were infected with 10⁶ PFU of VSV-ova and both the 1° and 2° effector CD8⁺ T cell populations were examined for expression of KLRG1, CD127, and CD62L (B).These data are representative of 2 independent experiments, each containing 4-5 mice per group.

Figure 8. Distinction recall potential and differentiation of secondary memory CD8⁺ T cell populations

Secondary memory CD8⁺ T cells (CD45.2⁺) were sorted into SLEC (KLRG1^high CD127^low), DPEC (KLRG1^high CD127^high), and MPEC (KLRG1^low CD127^high) to >98% purity and 10⁴ cells of each phenotype were adoptively transferred to naïve CD45.1⁺ mice. One day later those mice were challenged with 10⁴ CFU of LM-ova (A). The recall kinetics (B) and subsequent tertiary effector CD8⁺ T cell differentiation (C) of the Ova/K^b-specific memory CD8⁺ T cell sub-populations was tracked in the PBL using CD45.2 expression to identify the adoptively transferred cells and KLRG1 and CD127 expression to monitor effector cell differentiation. In the upper right corner of each plot is the mean percentage of the population in each quadrant. These data are representative of 2 independent experiments, each containing 4-5 mice per group.

Figure 9. Model of SLEC/MPEC differentiation under different inflammatory conditions

(A) The proportion of effector CD8⁺ T cells that differentiate into SLECs is highly dependent on the inflammatory milieu induced by infection or vaccination. Under conditions of high inflammation, such as after LM infection, SLEC differentiation is significantly enhanced by the expression of inflammatory cytokines, which include type I interferons (IFNα/β), Ebi3 (and therefore likely IL-27), IL-12 and IFNγ (top). In contrast, when only low levels of inflammation are induced, such as following DC vaccination (70), there is limited SLEC differentiation due to limited expression of inflammatory cytokines (bottom left). Mild inflammation results in fewer SLECs being generated, due to the limited expression of inflammatory cytokines. For example, during VSV infection type I interferons (IFNα/β) and Ebi3 are expressed, but IL-12 and IFNγare not expressed early. This inflammatory cytokine profile will result in significantly fewer SLECs being generated (middle). (B) ODN1826 treatment had significant effects on effector CD8⁺ T cell differentiation, which were either IL-12 –dependent or –independent. Enhancement of SLEC differentiation was highly dependent on IL-12, which is due to the ability of IL-12 to enhance T-bet expression (3). In contrast, enhancement of effector cytokine expression by ODN1826 treatment occurred through an IL-12-independent mechanism. Also, ODN1826 shifted the T_CM/T_EM ratio in favor of T_CM generation through an IL-12-independent mechanism.