Role of NKG2a/c+CD8+ T cells in pathogenic versus non-pathogenic SIV infections

Abstract

Some viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in the blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2_a/c ⁺CD8⁺ T cells increase in the blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2_a/c ⁺CD8⁺ T cells were not expanded in lymph nodes, and CXCR5⁺NKG2_a/c ⁺CD8⁺ T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2_a/c ⁺CD8⁺ T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, and immunoregulatory and gut barrier function, including CD73. NKG2_a/c ⁺CD8⁺ T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2_a/c ⁺CD8⁺ T cells in intestinal inflammation during SIV/HIV infections.

Keywords: Immunology; Transcriptomics; Viral Microbiology.

Graphical abstract

Figure 1

Identification and analysis of NKG2_a/c⁺ T cells in the blood of healthy nonhuman primates. Analyses were performed on six monkeys per species (A and B) Multidimensional viSNE analysis in blood cells of (A) AGM and (B) cynomolgus macaques (CM). Cells were stained with 16 markers and measured by flow cytometry. viSNE analysis was run on 60,000 live CD45⁺ single cells per sample using all surface markers. (C) Gating of the NKG2_a/c⁺ T cells based on CD45⁺ lin⁻CD3⁺NKG2_a/c⁺ cells. The numbers in each quadrant represent the percentage among CD45⁺ Lin⁻ cells in AGM (upper panel, blue) and CM (lower panel, orange). Each panel represents a distinct animal. Data from three representative animals per species are shown. (D) Mean fluorescence intensity (MFI) of NKG2_A/C at the surface of NKG2_a/c⁺ T cells, NK cells, and conventional CD8a⁺ T cells (left). Frequency of CD8a among NKG2_a/c⁺ T cells, NK cells, and conventional CD8a⁺ T cells (right). (E) Absolute numbers of NKG2_a/c⁺ T cells compared with NK and conventional CD8a⁺ T cells. (F) Frequencies of naive, memory, and effector NKG2_a/c⁺ T cells according to CD28 and CD95 expression. Dot plots with three representative animals per species are shown on the left. (G) MFI of intracellular cytokine expression ex vivo in unstimulated cells. (H) Frequency of cells expressing homing markers. (I) Frequencies of cells expressing NK cell markers. A nonparametric Mann-Whitney U test was applied; ∗p < 0.05, ∗∗p < 0.005. Horizontal bars indicate median values, and error bars indicate interquartile range. Each symbol (triangles for AGM and rectangles for CM) represents a distinct animal.

Figure 2

Dynamics of NKG2_a/c⁺CD8⁺ T cells in blood during pathogenic SIVmac infection and non-pathogenic SIVagm infection Nine animals per species were analyzed. AGM are represented in blue and CM in orange. (A) Dot plots illustrating the dynamics of CD45⁺CD20⁻CD3⁺NKG2_A/C⁺ cells during SIV infection in AGM (upper panel) and CM (lower panel). The days after SIV infection are indicated at the top of each dot plot. The dot plots show a representative animal for each species. The numbers in each quadrant indicate the percentage of NKG2_A/C⁺ T cells among CD45⁺ cells. (B) Frequency of CD8a⁺ cells among CD45⁺CD20⁻CD3⁺NKG2_A/C⁺ cells during SIV infection in CM (left) and AGM (right). (C) CD45⁺CD20⁻CD3⁺NKG2_A/C⁺CD8⁺ cell dynamics in response to SIV infection. The dark line represents the median. Day 0 represents the median value from all the time points analyzed before infection. (E–F) Dynamics of NKG2_a/c⁺CD8⁺ T cells expressing homing markers. The cells were followed in blood before and during the time course of SIV infection in nine AGM (blue) and nine CM (orange). Each symbol represents an individual monkey, the black line the median values, and day 0 the median values of all time points before infection. dpi: days post-infection. ∗p < 0.05.

Figure 3

Longitudinal analysis of NKG2a/c⁺ CD8⁺ T cell proliferation and functional phenotypes in blood and evaluation of correlation with viremia (A–D) Longitudinal analyses. Orange symbols indicate CM (left) and blue symbols AGM (right). Each symbol corresponds to a distinct animal. (A) Cell proliferation as evaluated by intracellular staining of Ki-67⁺. (B–D) Ex vivo suppressor activity as evaluated by (B) surface staining of CD107a and intracellular staining (C) of Perforin and (D) TNF-α. Each symbol represents a distinct animal. dpi: days post-infection. A nonparametric Mann-Whitney U test was applied, bars indicate median values, and error bars indicate interquartile range ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. (E–F) Correlation matrix of measures of viremia and NKG2a/c⁺ CD8⁺ T cell parameters. The Pearson correlation is shown. The matrix was built using the values obtained between day 0 and day 150 p.i from (E) 6 MAC and (F) 6 AGM.

Figure 4

Dynamics and functional phenotype of NKG2a/c⁺CD8⁺ T cells in secondary lymphoid organs and intestine from naive and SIV-infected monkeys Each symbol corresponds to a distinct animal. Orange symbols indicate CM, blue symbols AGM, violet symbols ART-treated CM, and red symbols rhesus macaques (RM). Bars indicate median values. (A) Frequencies of NKG2a/c⁺ CD8⁺ T cells among CD45⁺ cells in the spleen from CM (orange) and AGM (blue). (B) Dynamics of NKG2a/c⁺CD8⁺ T cells during SIV infection in peripheral lymph nodes (pLN) from CM (orange) and AGM (blue). The black line indicates the median values. (C) NKG2a/c⁺CD8⁺ T cells in the spleen of viremic and ART-treated virologically suppressed CM. (D) NKG2a/c⁺CD8⁺ T cells in the pLN of naive, viremic, and virological controller (SIC) RM. (E and F) Frequencies of NKG2a/c⁺CD8⁺ T cells expressing CXCR5 in (E) pLN and (F) spleen from CM (orange) and AGM (blue). (G) NKG2a/c⁺CD8⁺ T cells expressing CXCR5 in spleen from viremic and ART-treated virologically suppressed CM. (H) NKG2a/c⁺CD8⁺ T cells expressing CXCR5 in pLN from naive, viremic, and virological controller (SIC) RM. (I) Dynamics of NKG2a/c⁺CD8⁺ T cells in rectal biopsies from CM (orange) and AGM (blue). (J) Dynamics of NKG2a/c⁺ CD8⁺ T cells in distinct intestinal compartments from CM (orange) and AGM (blue). (K–M) Lymphocytes were isolated from rectal biopsies and surface (CD107a) or intracellular stainings (IFN-γ and Perforin) were assessed ex vivo in unstimulated NKG2a/c⁺CD8⁺ T cells for CM (orange) and AGM (blue). dpi: days post-infection. ∗p < 0.05.

Figure 5

Genome-wide transcriptome analysis of NKG2a/c⁺CD8⁺ T cells NKG2a/c⁺CD8⁺ T cells, conventional memory CD8⁺ T cells (CD95⁺CD28⁺), and conventional effector CD8⁺ T cells (CD95⁺CD28^-) were sorted from blood of five chronically SIVagm-infected AGM. (A and B) Heatmaps of unsupervised hierarchical clustering showing differentially expressed genes (A) between NKG2a/c⁺CD8⁺ T cells and conventional memory CD8⁺ T cells and (B) between NKG2a/c⁺CD8⁺ T cells and conventional effector CD8⁺ T cells. A p value adjustment was performed to take into account multiple testing and control the false-positive rate to a chosen level. The level of controlled false-positive rate was set to Padj <0.05. (C and D) Volcano plots that represent up- and down-regulated transcripts of NKG2a/c⁺CD8⁺ T cells when compared with (C) conventional memory CD8⁺ T cells (CD95⁺CD28⁺) (D) and conventional effector CD8⁺ T cells (CD95⁺CD28^-). The position of each data point is calculated as -log10 (p value) × log2 (FC), with a p value cutoff < 0.05. The red (up-regulated) and green dots (down-regulated) represent differentially expressed genes with a log2 fold change higher than 2. The list of all transcripts is given in Tables S2 and S3. (E–G) Graphs shows the enriched Gene Ontology (GO) terms for the genes that were (E) up-regulated in NKG2a/c⁺CD8⁺ T cells when compared with conventional memory CD8⁺ T cells (CD95⁺CD28⁺); (F) up-regulated in NKG2a/c⁺CD8⁺ T cells when compared with conventional effector CD8⁺ T cells (CD95⁺CD28⁺). No significant pathways were down-regulated when compared with conventional effector CD8⁺ T cells. The mRNAs were input for ClueGO plugged into Cytoscape for GO enrichment analysis. The “cellular component,” “biological process,” “molecular function,” and “immune system process” GO terms such as KEGG, Wiki pathways, and REACTOME were selected for this analysis. The two-sided hypergeometric test was used in the statistical inference. (G) Enriched GO terms for the genes that were down-regulated in NKG2a/c⁺CD8⁺ T cells when compared with conventional memory CD8⁺ T cells (CD95⁺CD28⁺). The adjusted p value threshold was set to 0.05. The term value corrected with the Bonferroni stepdown method was applied for p value correlation and indicated in the figure. Bars indicate the minus log of p-value. The list of the associated genes found for each term is given in Tables S4–S6.

Figure 6

Levels of systemic and intestinal inflammatory and cellular stress markers Each symbol corresponds to a distinct animal. Orange symbols indicate CM and blue symbols AGM. (A and B) Longitudinal IP-10 and Hsp60 concentrations in plasma during SIV infection in (A) CM and (B) AGM. (C and D) IP-10 and Hsp60 plasma concentrations were plotted against NKG2a/c⁺CD8⁺ T cell counts in blood of (C) CM and (D) AGM. (E and F) Longitudinal measurement of IL-23 and HSP60 concentrations produced by total rectal biopsy cells from (E) CM and (F) AGM. Horizontal bars indicate median values, and error bars indicate interquartile range. (G and H) IL-23 and HSP60 levels from rectal biopsies were plotted against NKG2a/c⁺CD8⁺ T cells of (C) CM and (D) AGM. NKG2a/c⁺CD8⁺ T cells corresponded to the percentage among CD45⁺ measured in the respective rectal biopsy. The Spearman correlation is shown, and r and p values are indicated in each graph. ∗p < 0.05.