KLRG1+ Memory CD8 T Cells Combine Properties of Short-Lived Effectors and Long-Lived Memory

Abstract

CD8 effector T cells with a CD127^hi KLRG1^- phenotype are considered precursors to the long-lived memory pool, whereas KLRG1⁺CD127^low cells are viewed as short-lived effectors. Nevertheless, we and others have shown that a KLRG1⁺CD127^low population persists into the memory phase and that these T cells (termed long-lived effector cells [LLEC]) display robust protective function during acute rechallenge with bacteria or viruses. Whether these T cells represent a true memory population or are instead a remnant effector cell population that failed to undergo initial contraction has remained unclear. In this study, we show that LLEC from mice express a distinct phenotypic and transcriptional signature that shares characteristics of both early effectors and long-lived memory cells. We also find that in contrast to KLRG1⁺ effector cells, LLEC undergo homeostatic proliferation and are not critically dependent on IL-15 for their maintenance. Furthermore, we find that LLEC are predominantly derived from KLRG1⁺ effector cells when isolated at day 12 of the response. Our work challenges the concept that the KLRG1⁺CD127^low population is dominated by short-lived cells and shows that KLRG1 downregulation is not a prerequisite to become a long-lived protective memory T cell.

Figure 1:. Long lived effector cells make up a distinct subset within the effector memory T cell compartment

(A) Ninety days post infection with LCMV-Arm, spleens were harvested from B6 mice and long-lived effector cells (LLEC) and effector memory cells (TEM) were differentiated based on their expression of CD27 and KLRG1. (B) Polyclonal LLEC have differing expression of several surface markers and transcription factors, including high expression of CX3CR1 and T-bet, and low expression of CD127, CXCR3, and Eomes when compared to TEM. C) LLEC were also identified within GP33-H2-Db tetramer positive cells 30 days post-infection with LCMV-Arm. D) CX3CR1 expression correlated with iv antibody labeling of LLEC in the spleen which is indicative of localization to the red pulp. E) LLEC and TEM OT-I memory cells were generated in mice challenged with virulent recombinant Listeria monocytogenes expressing ova (LM-OVA). At 90 days post-infection TEM and LLEC OT-I were FACS sorted and 2*10⁴ cells were transferred into congenially distinct naïve recipients. One day later, the recipient mice were challenged with LM-OVA and bacterial burden was determined in the spleen five days post challenge (F).

Figure 2:. LLEC maintain a unique transcriptional profile enriched for an effector affiliated gene signature

Splenic LLEC (CD44+ CD62L-KLRG1+ CD27-), TEM (CD44+ CD62L- KLRG1- CD27+), and TCM (CD44+CD62L+KLRG1-CD27+), and naïve CD8 T cells were FACS sorted from mice at least 90 days post infection with LCMV-Arm. Samples were mini-pooled with 3 mice per sample, resulting in 3 samples total. (A) Principal component analysis of all populations. (B) Hierarchical clustering depicting gene expression of the 75 most variable genes between memory subsets (LLEC, TEM, and TCM). C) t-distributed stochastic neighbor embedding (t-SNE) dimensional reduction was used to display unique clusters classified by a shared nearest neighbor algorithm (Seurat) via single cell sequencing (scRNA-seq) of the splenic P14 T cell population 60 days post LCMV-Arm infection. P14 cells were FACS sorted using Thy1.1 prior to sequencing. D) Gene expression levels of Klrg1, Cx3cr1, and Sell visualized by color intensity (t-SNE plot) and violin plots.

Figure 3:. Upregulation of memory associated genes differentiates LLEC from short lived precursors

(A) CD122, CD127, CD25, Eomes, and T-bet protein expression was compared in KLRG1+ CD27- and KLRG1- CD27+ P14 CD8 T cells were compared 12 days and 100 days post LCMV-Arm infection, with representative staining shown in (A) and quantification shown in (B). (C) Gene set enrichment analysis (GSEA) comparing genes differentially expressed in LLEC vs TEM to genes upregulated in short-lived effector CD8 T cells compared to memory precursor cells (GSE8678) reveals enrichment for short-lived effector transcriptional phenotype. (D) Gene set enrichment analysis (GSEA) comparing genes differentially expressed in LLEC vs TEM to genes downregulated in short-lived effector CD8 T cells compared to memory precursor cells (GSE8678) reveals enrichment for short-lived effector transcriptional phenotype. (E) Volcano plot showing gene differential expression by fold change and p-value. LLEC and TEM gene list generated in Fig 2. (F) Heatmap comparing differential gene expression of hallmark memory genes between TCM, TEM, LLEC, and naïve t cells. (G) qPCR confirmation of Dmrta1, Prss12, and Aqp9 expression in T cell subsets.

Figure 4:. Effector cell gene expression is reduced in LLEC compared to SLEC

A) P14 cells were transferred to C57Bl/6 mice and challenged with LCMV-Arm. At 12 days post-infection(dpi) and 60 dpi P14 cells were isolated from the spleen and processed for scRNA-seq using the 10X Chromium platform (10X Genomics). Datasets from 12dpi and 60dpi were aligned and merged using the Seurat R package. Following PCA, t-distributed stochastic neighbor embedding (t-SNE) dimensional reduction was completed and cells were labeled based on clustering using Seurat. B) t-SNE plot of aligned merged P14 data sets reveals family of cells defined by effector associated expression profile. C) Expression distribution of Klrg1, Cx3cr1, Il7r, and Gzma by cells. D) Proportion of total cells at day 12 and day 60 post infection timepoints that cluster within the ‘effector-like’ merged cluster. (E). Differential gene expression analysis comparing day 12 and day 60 cells (from only the effector cell cluster) resulted in 85 DE genes. Unsupervised hierarchical clustering of these genes with the scaled dataset highlights the gene expression differences between the two timepoints. F) Split dot plots comparing differentially expressed genes between day 12 and day 60 cells within effector-like cell cluster. Size of dots represents frequency of cells within the cluster expressing and the intensity of the color indicates the expression level of the indicated gene.

Figure 5:. The IL15 dependency of LLEC is similar to other memory cells, but homeostatic turnover is low.

A) Congenically labelled P14 cells were isolated from LCMV-Armstrong challenged mice. D14 effector cells (CD45.1/1) and D30 memory cells (CD45.½) were transferred into C57BL/6 or IL15-KO recipients (CD45.2/2). B) 10 days post transfer spleens were harvested for flow cytometry. C) Donor P14 cells were analyzed for phenotype of surviving cells. P14 memory cells had similar frequencies of KLRG1+ cells in both C57BL/6 and IL15 knockout recipients. Effector P14 cells transferred to IL15 knockout recipients had reduced KLRG1+ cells relative to those transferred to C57BL/6 recipients. n=3 mice, representative from two independent experiments D) Memory P14 cells were established by transferring naïve P14 cells and challenging mice with LCMV-Armstrong infection. 30 days post infection mice were treated with BrdU in drinking water for two weeks. 14 days post transfer BrdU incorporation into donor cells was quantified by flow cytometry. n=4 mice, representative of two independent experiments.

Figure 6:. LLEC are derived from KLRG1+ short-lived effector cells

P14 CD8 T cells were transferred into congenically distinct recipients. One day later, the recipients were infected with LCMV-Arm. (A) On day 12 post infection, P14 cells were FACS sorted based on KLRG1 and CD127 expression (A). These populations were transferred into naïve recipients. Thirty days post transfer, mice were harvested and donor cells enriched by tetramer pulldown. (B) Thirty days post transfer the Day 12 KLRG1- CD127+ (MPEC) CD8 T cells produced more long-lived cells than KLRG1+CD127- (SLEC) and KLRG1+CD127+ precursors. (C) Representative flow cytometry plots depicting donor cell phenotype at day 30 post transfer. (D) Frequency and number of TCM (CD44⁺ CD62L⁺), TEM (CD44⁺ CD62L^- KLRG1^- CD27⁺), and LLEC (CD44⁺ CD62L^- KLRG1⁺ CD27^-) phenotype transferred cells.

Figure 7.. KLRG1+ effectors preferentially generate LLEC

A) Naïve P14 CD8 T cells were transferred to congenically distinct recipients and one day later infected with LCMV-Arm. 4 days post infection, P14 cells were sorted based on KLRG1 expression and transferred into LCMV-Arm infection matched recipients. Thirty days post transfer donor cells were enriched by tetramer pulldown and analyzed by flow cytometry. B) The total number of P14 memory cells generated from KLRG1^hi and KLRG1^low donor cells. Frequency (C) and number (D) of TCM (CD44⁺ CD62L⁺), TEM (CD44⁺ CD62L^- KLRG1^- CD27⁺), and LLEC (CD44⁺ CD62L^- KLRG1⁺ CD27^-) phenotype cells. E) gMFI of KLRG1, GP33-Tetramer, CX3CR1, and CD127 of LLEC that resulted from either KLRG1^hi and KLRG1^low donor cells.