Circulating KLRG1+ long-lived effector memory T cells retain the flexibility to become tissue resident

Abstract

KLRG1⁺ CD8 T cells persist for months after clearance of acute infections and maintain high levels of effector molecules, contributing protective immunity against systemic pathogens. Upon secondary infection, these long-lived effector cells (LLECs) are incapable of forming other circulating KLRG1^- memory subsets such as central and effector memory T cells. Thus, KLRG1⁺ memory T cells are frequently referred to as a terminally differentiated population that is relatively short lived. Here, we show that after viral infection of mice, effector cells derived from LLECs rapidly enter nonlymphoid tissues and reduce pathogen burden but are largely dependent on receiving antigen cues from vascular endothelial cells. Single-cell RNA sequencing reveals that secondary memory cells in nonlymphoid tissues arising from either KLRG1⁺ or KLRG1^- memory precursors develop a similar resident memory transcriptional signature. Thus, although LLECs cannot differentiate into other circulating memory populations, they still retain the flexibility to enter tissues and establish residency.

Fig. 1.. KLRG1⁺ LLECs are restricted to the bloodstream and spleen.

(A) Experimental outline for (B) to (D). (B) Representative flow plots showing the P14 CD8 memory subsets in blood, lymphoid, and nonlymphoid tissues after LCMV infection. The quadrant labeled in red identifies LLECs. (C) Representative flow plots showing P14 CD8 memory subsets in IV⁺ and IV⁻ lung tissue after IAV-GP33 infection. (D) Representative histograms of surface marker expression on different P14 CD8 memory subsets. (E) Experimental outline for (F) and (G). (F) Representative flow plots of the P14 CD8 memory subsets from donor P14 TCM cells, TEM cells, and LLECs after challenge with LCMV. The quadrants labeled in red identifies LLECs. (G) Quantification of (E) showing the percentage of donor P14 memory cells in memory subsets. All experiments were repeated at least twice with four or five mice per group. Data shown are pooled from two separate experiments, and error bars display SEM.

Fig. 2.. LLEC-derived cells enter the tissue during LCMV challenge.

(A) Representative flow plots showing the frequency of transferred P14 TCM cells, TEM cells, and LLECs in Vα2⁺ CD8 T cells from the spleen 9 days after LCMV infection. (B) Quantification of (A) showing the frequency and number of transferred memory P14 cells in the spleen. (C) Representative flow plots showing the frequency of transferred TCM, TEM, and LLEC P14 cells among Vα2⁺ CD8 T cells from the kidney. (D) Quantification of the number of transferred TCM cell–, TEM cell–, and LLEC-derived P14 T cells in the kidney, SI-IEL, and salivary gland 9 days after LCMV infection. (E) Representative immunofluorescence microscopy images of the small intestine and salivary gland 9 days after LCMV infection. DAPI (cyan), CD31 (white), CD45.1 (yellow), and CD8 (red) are shown. White arrowheads indicate transferred cells found in the tissue parenchyma, and white arrows indicate endogenous T cells. Images are ×200 magnification; scale bars, 75 μm. (F) Representative flow plots of transferred endogenous, memory-derived, tetramer⁺ cells in the liver 9 days after LCMV infection. (G) Quantification of the number of transferred gp33⁺ cells in the liver and kidney. (H) Representative flow plots and quantification of LLEC and TEM cell division after cotransfer. (I) Representative histograms showing KLRG1 staining on transferred TCM cell–, TEM cell–, and LLEC-derived P14 cells. (J) Frequency of KLRG1⁺ P14 cells derived from transferred memory cells in the kidney, SI-IEL, and SG. All experiments were repeated at least twice with four or five mice per group. All error bars shown are SEM. Data shown in (B), (D), (G), and (J) are from one experiment. Data shown in (H) are combined from two experiments. For (B), (D), and (J), one-way ANOVAs followed by Tukey’s multiple comparisons test were used. For (G) and (H), unpaired, two-tailed Student’s t tests were used.

Fig. 3.. LLEC-derived cells enter the lung after flu infection with an intact memory compartment.

(A) Representative flow plots showing transferred TCM cell–, TEM cell–, and LLEC-derived P14 cells in the lung after flu infection. (B) Quantification of (A) showing the number of TCM cells, TEM cells, and LLEC-derived P14 cells in the lung tissue (IV⁻) or lung vasculature (IV⁺). (C) Quantification of the number of TCM cell–, TEM cell–, and LLEC-derived P14 cells in the LN. (D) Frequency of Ki67⁺ TCM cell– or LLEC-derived cells in the lung. (E) Quantification of PFUs per lung 5 days after infection with IAV-gp33. (F) Experimental outline for (G) to (K). (G) Representative flow plot and quantification of CX3CR1-YFP reporter expression in the bloodstream of mice before flu infection. (H) Representative flow plot showing KLRG1 by CD62L expression on CX3CR1-YFP⁺ P14 CD8 T cells in the blood after flu infection. (I) Quantification of (H) showing memory subsets on CX3CR1-YFP⁺ P14 CD8 T cells. (J) Representative flow plots of IV⁻ and IV⁺ CX3CR1-YFP P14 cells in the lung after flu infection. (K) Quantification of IV⁻ and IV⁺ CX3CR1-YFP P14 T cells in the lung after flu infection. All experiments were repeated at least twice with four or five mice per group. Data shown are from one experiment. All error bars shown are SEM. For (B), (C), (E), (I), and (K), one-way ANOVA followed by Tukey’s multiple comparisons test was used. For (D), an unpaired, two-tailed Student’s t test was used.

Fig. 4.. LLECs enter the tissue parenchyma earlier than TCM cells via antigen presentation on endothelial cells.

(A) Experimental outline for (B) and (C). (B) Representative flow plots showing transferred LLECs and TCM P14 cells in the spleen and lung tissue at 3 days after IAV-gp33 infection. (C) Quantification of the ratio of transferred LLECs to TCM cells in the spleen and lung. (D) Experimental outline for (E) to (H). (E) Representative flow plot of MHC class I expression on CD31⁺ endothelial cells and CD45⁺ leukocytes in tamoxifen-treated Cre⁺ and Cre⁻ mice. (F) Quantification of MHC class I MFI. (G) Representative flow plots of transferred LLEC and TCM P14 cells in the lungs of Cre⁻ and Cre⁺ mice. (H) Quantification of the number of transferred LLEC and TCM P14 cells in the lung. Experiments were repeated twice. Data in (C) and (H) are from two experiments with three mice per group in each experiment. Data in (F) are from one experiment. All error bars shown are SEM. For (C), a paired, two-tailed Student’s t test was used. For (F), an unpaired, two-tailed Student’s t test was used. One-way ANOVA followed by Tukey’s multiple comparisons test were used in (H).

Fig. 5.. LLEC-derived cells are found in nonlymphoid tissue at memory after infectious challenge.

(A) Representative flow plots showing transferred TCM cell–, TEM cell–, and LLEC-derived P14 T cells in the kidney after LCMV infection. (B) Quantification of the number of transferred TCM cell–, TEM cell–, and LLEC-derived P14 T cells in the kidney and SI-IEL. (C) Representative flow plots showing KLRG1 and CX3CR1 expression on LLEC-derived P14 cells in the blood and SI-IEL. (D) Quantification of (C) showing the frequency of KLRG1⁺CX3CR1⁺ double-positive cells in the blood, kidney, and SI-IEL. (E) Representative flow plots showing CD69 and CD103 expression on transferred TCM cell–, TEM cell–, and LLEC-derived P14 T cells in the kidney and SI-IEL. (F) Quantification of (E) showing the frequency of CD69⁺ and CD103⁺ transferred TCM cell–, TEM cell–, and LLEC-derived P14 T cells in the SI-IEL. (G) Quantification of (E) showing the frequency of CD69⁺ transferred TCM cell–, TEM cell–, and LLEC-derived P14 T cells in the kidney. (H) Representative flow plots showing transferred TCM cell– and LLEC-derived P14 T cells in the lung after IAV-gp33 infection. (I) Quantification of (H) showing the number of transferred TCM cell– and LLEC-derived P14 T cells per lung. (J) Representative flow plots showing CD69 and CD103 expression on transferred TCM cell– and LLEC-derived P14 T cells in the lung. (K) Quantification of (J) showing the frequency of CD69⁺ and CD103⁺ transferred TCM cell– and LLEC-derived P14 T cells in the lung. All experiments were repeated at least twice with four or five mice per group. Data in (B), (F), and (G) are pooled from two separate experiments, whereas data in (D), (I), and (K) are from one experiment. All error bars shown are SEM. For (B), (D), (F), and (G), one-way ANOVA followed by Tukey’s multiple comparisons test were used. For (I) and (K), unpaired, two-tailed Student’s t tests were used.

Fig. 6.. LLEC-derived cells become TRM cells in nonlymphoid tissue.

(A) UMAP representation of cells. Individual clusters labeled. (B) UMAP representation of cells. Cells labeled by cell line and tissue type. (C) UMAP representations of cells separated by tissue and donor cell type. (D) Frequency of each donor cell type making up each cluster in individual tissues. (E) Average expression of TRM cell signature genes and average expression of Tcirc cell signature genes. (F) Heatmap of average gene expression by cluster and cell type for select genes. Values are scaled by row. (G) KLRG1 protein and CX3CR1 protein expression. (H) CD69 protein and CD103 protein expression. Data are from one CITE-seq single-cell gene and protein expression experiment. Values in (A) to (F) are based on normalized gene expression values. Values in (G) are based on normalized protein expression values.