Environmental cues dictate the fate of individual CD8+ T cells responding to infection

Abstract

Many studies have examined pathways controlling effector T cell differentiation, but less is known about the fate of individual CD8+ T cells during infection. Here, we examine the antiviral and antibacterial responses of single CD8+ T cells from the polyclonal repertoire. The progeny of naive clonal CD8+ T cells displayed unique profiles of differentiation based on extrinsic pathogen-induced environmental cues, with some clones demonstrating extreme bias toward a single developmental pathway. Moreover, even within the same animal, a single naive CD8+ T cell exhibited distinct fates that were controlled by tissue-specific events. However, memory CD8+ T cells relied on intrinsic factors to control differentiation upon challenge. Our results demonstrate that stochastic and instructive events differentially contribute to shaping the primary and secondary CD8+ T cell response and provide insight into the underlying forces that drive effector differentiation and protective memory formation.

Figure 1

Detection and phenotype of the progeny of single antigen-specific CD8⁺ T cells in response to viral infection. OVA-specific CD8⁺ T cells from the spleen were identified by OVAK^b tetramer staining, and donor populations were identified by CD45.1 and CD45.2 staining within the OVA-K^b+ population. Expression of KLRG1 and CD127 was then analyzed on gated donor populations. This experiment was repeated 3 times, with 60 mice total.

Figure 2

Pathogen-specific effector subset development from CD8⁺ T cell clones. a. Stacked graphs of the phenotypes of the progeny of 20 individual OVA-specific clones observed from one VSV-OVA experiment (20 mice, 40 possible clones). This experiment was repeated 4 times, with 80 mice total. b. Stacked graphs of the progeny of 17 individual OVA-specific clones from one LM-OVA experiment (20 mice, 40 possible clones). This experiment was repeated 3 times, with 60 mice total. c. 9 examples of distinct phenotypes of donor populations after VSV-OVA infection. Clones were classified as MPEC phenotype (2-fold greater MPEC than SLEC), SLEC phenotype (2-fold greater SLEC than MPEC), or MIXED. See also Figure S2.

Figure 3

Concatenation of responding clonal progeny recapitulates the host polyclonal CD8⁺ T cell response to infection. a. Phenotypes as classified in Fig. 2c., of all OVA- and N-specific clones from VSV-OVA and LM-OVA experiments. VSV-OVA and VSV-N clones are from 80 mice total (4 separate experiments) with 160 possible clones each. LM-OVA clones are from 60 mice (3 separate experiments) with 120 possible clones. b. The number of cells of MPEC, SLEC, DEPC or EEC phenotype for all clones was determined then mathematically combined to calculate total frequency of each effector phenotype in all VSV-OVA clones, VSV-N clones and LM-OVA clones. c. Breakdown of effector phenotypes within the bulk host OVA- and N-specific response in the same mice from part b. See also Figure S3.

Figure 4

The fate of a single clone is distinct between the spleen and the intestinal mucosa. a. Stacked graphs of the phenotype of the progeny of 9 transferred OVA-specific clones (present in 20 mice) seven days after VSV-OVA infection in both spleen (S) and peripheral LN (L). Peripheral lymph nodes include axial, brachial, and inguinal. b. Stacked graphs of the phenotype of the progeny of 8 transferred OVA-specific clones found 10 days after oral LM-OVA infection in both the spleen (S) and intestinal epithelium (I). The first set of stacked graphs represents the average distribution of effector populations in the bulk host response of all 20 mice in each experiment. This experiment was repeated twice with similar results.

Figure 5

Biased development toward KLRG1⁺ secondary effector cells is intrinsic to memory CD8⁺ T cells. a. Phenotype of polyclonal OVA-specific memory CD8⁺ T cells after VSV-OVA infection (>60 days). b. One day after transfer of bulk VSV-OVA-specific memory cells into naïve recipients, mice were infected with VSV-OVA. Phenotype of transferred OVA-specific memory cells 6 days later is shown. c. A single CD45.1 OVA-specific memory CD8⁺ T cell and a single CD45.1/2 OVA-specific naïve CD8⁺ T cell were transferred to naïve mice that were then infected with VSV-OVA. Seven days later KLRG1 and CD127 expression was analyzed on the donor populations. d. Graphs show the phenotype of 13 clones derived from single memory cells and 10 clones derived from naïve cells from one VSV-OVA experiment (20 mice). This experiment was repeated 4 times with 80 mice total.

Figure 6

Clonal burst size is divergent between different specificities and endogenous compared to transgenic CD8⁺ T cells. a. Total number of cells present in the spleen derived from each transferred clone for all experimental conditions as indicated. Mean clone size values are shown with red bars. Below is shown a table of mean and median clone size for all conditions. Two stars indicate a p-value of less than 0.005 and three stars indicate a p-value of less than 0.0005 based on an unpaired T-test. Burst sizes of progeny of OVA-specific (b.) and N-specific clones (c.) from VSV-OVA infected recipients based on the percentage of KLRG1⁺ CD127- cells within each clone. Burst sizes of progeny of OVA-specific clones from LM-OVA infected recipients (d.) and OVA-specific clones from VSV-infected recipients following single OT-I transfer (e.). Spearman correlation (r) and p-values are displayed on each graph. These data are compiled from all of the experiments shown in Figures 2,3 and 5.