Phenotypic and Functional Alterations in Circulating Memory CD8 T Cells with Time after Primary Infection

<div><p>Memory CD8 T cells confer increased protection to immune hosts upon secondary viral, bacterial, and parasitic infections. The level of protection provided depends on the numbers, quality (functional ability), and location of memory CD8 T cells present at the time of infection. While primary memory CD8 T cells can be maintained for the life of the host, the full extent of phenotypic and functional changes that occur over time after initial antigen encounter remains poorly characterized. Here we show that critical properties of circulating primary memory CD8 T cells, including location, phenotype, cytokine production, maintenance, secondary proliferation, secondary memory generation potential, and mitochondrial function change with time after infection. Interestingly, phenotypic and functional alterations in the memory population are not due solely to shifts in the ratio of effector (CD62Llo) and central memory (CD62Lhi) cells, but also occur within defined CD62Lhi memory CD8 T cell subsets. CD62Lhi memory cells retain the ability to efficiently produce cytokines with time after infection. However, while it is was not formally tested whether changes in CD62Lhi memory CD8 T cells over time occur in a cell intrinsic manner or are due to selective death and/or survival, the gene expression profiles of CD62Lhi memory CD8 T cells change, phenotypic heterogeneity decreases, and mitochondrial function and proliferative capacity in either a lymphopenic environment or in response to antigen re-encounter increase with time. Importantly, and in accordance with their enhanced proliferative and metabolic capabilities, protection provided against chronic LCMV clone-13 infection increases over time for both circulating memory CD8 T cell populations and for CD62Lhi memory cells. Taken together, the data in this study reveal that memory CD8 T cells continue to change with time after infection and suggest that the outcome of vaccination strategies designed to elicit protective memory CD8 T cells using single or prime-boost immunizations depends upon the timing between antigen encounters.</p></div

Phenotypic heterogeneity of memory CD8 T cells decreases with time after infection.

<p><i>early</i>M (30–45 days p.i.) and <i>late</i>M (8+ months p.i.) P14 cells from the indicated organs were co-stained for CD62L, CD27, CD122, CD127, KLRG1, and CD11b. (A) Representative histograms of CD27, CD127, and CD122 expression on gated CD62Lhi <i>early</i>M (open histogram) and <i>late</i>M (grey histogram) P14 cells in the indicated organs. Numbers inside histograms indicate the percentage of CD62Lhi <i>early</i>M (top) or <i>late</i>M (bottom) cells staining positive for the indicated marker. (B) Number of subpopulations (out of 32 possible) comprising greater than 1% of the total CD62Lhi P14 population for <i>early</i>M and <i>late</i>M P14 cells in the indicated organs. Representative data from one of three individual experiments with 3 mice per group per experiment. Error bars represent the standard error of the mean.</p

Localization, phenotype, and function of memory CD8 T cells changes with time after infection.

<p>Memory P14 cells were generated by adoptively transferring 5x10³ naïve P14 T cells into Thy disparate naïve recipients followed by i.p. injection of LCMV 24 hours later. Analysis was performed on <i>early</i>M (30–45 days p.i.) and <i>late</i>M (8+ months p.i.) P14 cells. (A) Numbers of <i>early</i>M and <i>late</i>M P14 cells found in the spleens of recipient mice. (B) Ratios of the percentage of <i>early</i>M or <i>late</i>M P14 cells in the indicated organs following perfusion of tissues out of total lymphocytes to the percentage of <i>early</i>M or <i>late</i>M P14 cells in PBL out of total lymphocytes. (C) Representative histograms showing CD127, CD62L, CD27, CD122, and KLRG1 expression on gated <i>early</i>M (open histograms) and <i>late</i>M (grey histograms) P14 cells isolated from spleens. (D) Percentage of gated <i>early</i>M or <i>late</i>M P14 cells producing IFN-γ, TNF-α, or IL-2 as measured by ICS, or undergoing degranulation as measured by surface CD107a expression following 5hr incubation with GP_33-41 peptide. (E) Percentage of gated <i>early</i>M or <i>late</i>M P14 cells producing one (IFN-γ), two (IFN-γ and TNF-α), or three (IFN-γ, TNF-α, and IL-2) cytokines as measured by ICS following 5hr incubation with GP_33-41 peptide. (F) Representative dot plots of BrdU staining for gated <i>early</i>M or <i>late</i>M P14 cells in PBL 8 days after BrdU injection. (G) Representative histograms of CFSE dilution on gated <i>early</i>M (open histograms) or <i>late</i>M (grey histograms) P14 cells after 3 day culture in the presence or absence (black histograms) of the indicated concentrations of IL-15. NS, not statistically significant; *, statistically significant (p<0.05); **, statistically significant (p<0.01) as determined by Student t-test. Representative data from one of three individual experiments with 3 mice per group per experiment. Error bars represent the standard error of the mean.</p

Cytokine production, degranulation, and functional avidity, of CD62Lhi memory are not influenced by time.

<p>Analysis was performed on CD62Lhi <i>early</i>M (30–45 days p.i.) and <i>late</i>M (8+ months p.i.) P14 cells. (A) Representative histograms from gated CD62L+ <i>early</i>M or <i>late</i>M P14 cells showing IFN-γ, TNF-α, or IL-2 production as measured by ICS, or surface CD107a expression as a measure of degranulation following 5hr incubation with GP_33-41 peptide. (B) Percentage of gated CD62Lhi <i>early</i>M or <i>late</i>M P14 cells producing IFN-γ, TNF-α, or IL-2 or undergoing degranulation. (C) Functional avidity curves for CD62Lhi <i>early</i>M (open squares) and <i>late</i>M (black squares) P14 cells was determined by ICS following 5hr incubation of cells with the indicated concentrations of GP_33-41 peptide. (D) EC50 for CD62Lhi <i>early</i>M and <i>late</i>M P14 cells as determined from functional avidity curves. (E) Time required for CD62Lhi <i>early</i>M (open squares) and <i>late</i>M (black squares) P14 cells to produce IFN-γ, TNF-α, or IL-2 was determined by ICS following incubation with GP_33-41 peptide for the indicated lengths of time. NS, not statistically significant as determined by Student t-test. Representative data from one of three individual experiments with 3 mice per group per experiment. Error bars represent the standard error of the mean.</p

<i>late</i>M cells provide better protection than <i>early</i>M cells following LCMV clone-13 infection.

<p>(A-D) Sorted CD62Lhi <i>early</i>M (30–45 days p.i.) or <i>late</i>M (8+ months p.i.) P14 cells were injected into naïve recipients followed by i.v. injection of LM or LCMV clone-13 24 hours later. (E-F) Positively selected <i>early</i>M (30–45 days p.i.) or <i>late</i>M (8+ months p.i.) populations of P14 cells were injected into naïve recipients followed by i.v. injection of LM or LCMV clone-13 24 hours later. (A and E) Bacterial CFUs in the spleen were measured three days after LM infection for mice that received no cells (naïve), or adoptive transfer of 7x10⁴ sorted CD62Lhi <i>early</i>M or <i>late</i>M P14 cells (A) or 7x10⁴ positively selected <i>early</i>M or <i>late</i>M P14 cell populations (E). (B and F) Viral PFUs in the spleen were measured 8 days after LCMV clone-13 infection for mice that received no cells (naïve), or adoptive transfer of 5x10⁴ sorted CD62Lhi <i>early</i>M or <i>late</i>M P14 cells (B) or 7x10⁴ positively selected <i>early</i>M or <i>late</i>M P14 cell populations (F). (C and G) Kinetic analysis of secondary responses generated from sorted CD62Lhi <i>early</i>M (open squares) or <i>late</i>M (black squares) P14 cells (C) and secondary responses generated from positively selected <i>early</i>M (open squares) or <i>late</i>M (black squares) P14 cell populations (G) in PBL at the indicated days post LCMV clone-13 infection. (D and H) Numbers of progeny P14 cells generated from transferred CD62Lhi <i>early</i>M or <i>late</i>M cells (D) and transferred <i>early</i>M or <i>late</i>M cell populations (H) found in the spleens of recipient mice 8 days post LCMV clone-13 infection. NS, not statistically significant; *, statistically significant (p<0.05) as determined by Student t-test or ANOVA with a Bonferroni post-test. Representative data from one of two to three individual experiments with 3 mice per group per experiment. Error bars represent the standard error of the mean.</p

Gene expression patterns among CD62Lhi memory CD8 T cells change with time after infection.

<p>mRNA was isolated from sorted CD62Lhi <i>early</i>M (30–45 days p.i.) and <i>late</i>M (8+ months p.i.) P14 cells and used for microarray hybridization. (A) Heat map of genes with significantly different mRNA expression between <i>early</i>M and <i>late</i>M CD62Lhi P14 cells with a fold change >1.25. (B) Biological pathway analysis of genes with significant mRNA changes of fold >1.25 in CD62Lhi <i>early</i>M and <i>late</i>M P14 cells was generated using the KEGG pathway tool in DAVID bioinformatics resources. ↑, genes with increased expression in CD62Lhi <i>late</i>M compared to <i>early</i>M P14 cells. ↓, genes with decreased expression in CD62Lhi <i>late</i>M compared to <i>early</i>M P14 cells. Data were generated from six separate microarray hybridizations performed using mRNA extracted from sorted <i>early</i>M and <i>late</i>M P14 cells from three individual mice per group.</p

Mitochondrial function of memory CD8 T cells increases with time after infection.

<p>(A) Biological pathway analysis based on microarray data of genes with significant mRNA changes of fold >1.25 in CD62Lhi <i>early</i>M and <i>late</i>M P14 cells was generated using the KEGG pathway tool in DAVID bioinformatics resources. Shown is relative gene expression for genes involved in the KEGG oxidative phosphorylation pathway that are differentially expressed between CD62Lhi <i>early</i>M and <i>late</i>M P14 cells. Positive fold changes represent genes with increased expression in CD62Lhi <i>late</i>M compared to <i>early</i>M P14 cells while negative fold changes represent genes with decreased expression in CD62Lhi <i>late</i>M compared to <i>early</i>M P14 cells. (B) Gene set enrichment analysis was performed comparing expression of genes in CD62Lhi <i>early</i>M and <i>late</i>M P14 cells to existing gene sets. <i>late</i>M cells showed enrichment for oxidative phosphorylation gene sets. (C-F) <i>early</i>M (30–45 days p.i.) and <i>late</i>M (8+ months p.i.) populations or CD62Lhi P14 cells were purified and 2x10⁵ cells were plated in XF media and analyzed with an XF-96 extracellular flux analyzer (Seahorse Bioscience). (C) Ratio of O₂ consumption rates (OCR) to extracellular acidification rates (ECAR) measured under basal conditions for CD62Lhi <i>early</i>M or <i>late</i>M P14 cells. (D) OCR was measured under basal conditions and in response to the indicated mitochondrial inhibitors for <i>early</i>M or <i>late</i>M P14 cell populations. (E) Ratio of OCR to ECAR measured under basal conditions for <i>early</i>M or <i>late</i>M P14 cell populations. (F) Spare respiratory capacity (SRC) of <i>early</i>M or <i>late</i>M P14 cell populations as indicated by maximum OCR following FCCP injection as a percentage of maximum OCR under basal conditions. NS, not statistically significant; *, statistically significant (p<0.05); **, statistically significant (p<0.01) as determined by Student t-test. Combined data from four individual experiments that provided similar results with 3 mice per group per experiment. Error bars represent the standard error of the mean.</p

Proliferation and ‘memory generation potential’ of CD62Lhi memory cells increases with time after infection.

<p>Sorted CD62Lhi Thy disparate <i>early</i>M and <i>late</i>M P14 cells were mixed and injected into naïve recipients followed by i.p. injection of LCMV Armstrong 24 hours later. (A) Dot plot showing the master mix of CD62Lhi <i>early</i>M (Thy1.1/1.1) and <i>late</i>M (Thy1.1/1.2) P14 cells used for adoptive transfer. (B) Representative plot showing the response of progeny of sorted CD62Lhi <i>early</i>M (Thy1.1/1.1) and <i>late</i>M (Thy1.1/1.2) P14 cells in PBL d8 after LCMV infection. (C) Kinetic analysis of secondary responses generated from sorted CD62Lhi <i>early</i>M (open squares) and <i>late</i>M (black squares) P14 cells in PBL at the indicated days post LCMV infection. (D) Ratio of cells generated from sorted CD62Lhi <i>late</i>M to <i>early</i>M P14 cells in PBL at the indicated days post LCMV infection. Doted line indicates the starting ratio of CD62Lhi <i>late</i>M to <i>early</i>M P14 cells. (E) Ratio of cells generated from sorted CD62Lhi <i>late</i>M to <i>early</i>M P14 cells in the indicated organs at a memory time point (d140) following LCMV infection. Doted line indicates the starting ratio of CD62Lhi <i>late</i>M to <i>early</i>M P14 cells. R:, ratio of CD62Lhi <i>late</i>M to <i>early</i>M P14 cells or progeny of CD62Lhi <i>late</i>M to <i>early</i>M P14 cells.*, statistically significant (p<0.05) as determined by Student t-test. Representative data from one of two individual experiments with 5 mice per group per experiment. Error bars represent the standard error of the mean.</p

Sensitivity to IL-15 and ability to undergo homeostatic proliferation for CD62Lhi memory changes with time.

<p>Analysis was performed on CD62Lhi <i>early</i>M (30–45 days p.i.) and <i>late</i>M (8+ months p.i.) P14 cells. (A) A comparison of relative mRNA expression of the indicated components of the IL-15R complex between CD62Lhi <i>early</i>M and <i>late</i>M P14 cells from microarray data. Positive fold changes represent genes with increased expression in CD62Lhi <i>late</i>M compared to <i>early</i>M P14 cells. (B) Representative histograms of CD122 expression on gated CD62Lhi <i>early</i>M (open histogram) and <i>late</i>M (grey histogram) P14 cells. (C) Representative histograms of CFSE dilution on gated CD62Lhi <i>early</i>M (open histograms) or <i>late</i>M (grey histograms) P14 cells after 3 day culture in the presence or absence (black histograms) of the indicated concentrations of IL-15. (D) CD62Lhi <i>early</i>M and <i>late</i>M P14 cells were sorted and co-transferred into Rag-/- mice. <i>Left</i>. Dot plot showing the master mix of <i>early</i>M (Thy1.1/1.2) and <i>late</i>M (Thy1.1/1.1) P14 cells used for adoptive transfer. <i>Right</i>. Representative dot plot of transferred CD62Lhi <i>early</i>M (Thy1.1/1.2) and <i>late</i>M (Thy1.1/1.1) P14 cells isolated from spleens of Rag-/- mice d15 after adoptive transfer. R:, ratio of CD62Lhi <i>late</i>M to <i>early</i>M P14 cells. **, statistically significant (p<0.01) as determined by Student t-test.</p

Functional annotation of genes significantly different between CD62Lhi <i>early</i>M and <i>late</i>M P14 cells at fold >1.5.

<p>Positive fold changes indicate increased expression in <i>late</i>M compared to <i>early</i>M CD62Lhi P14 cells</p><p>Functional annotation of genes significantly different between CD62Lhi <i>early</i>M and <i>late</i>M P14 cells at fold >1.5.</p