Loss of circulating CD8α+ NK cells during human Mycobacterium tuberculosis infection

Abstract

Natural Killer (NK) cells can recognize and kill Mtb-infected cells in vitro, however their role after natural human exposure has not been well-studied. To identify Mtb-responsive NK cell populations, we analyzed the peripheral blood of healthy household contacts of active Tuberculosis (TB) cases and source community donors in an endemic region of Port-au-Prince, Haiti by flow cytometry. We observed higher CD8α expression on NK cells in putative resistors (IGRA- contacts) with a progressive loss of these circulating cells during household-associated latent infection and disease. In vitro assays and CITE-seq analysis of CD8α⁺ NK cells demonstrated enhanced maturity, cytotoxic gene expression, and response to cytokine stimulation relative to CD8α^- NK cells. CD8α⁺ NK cells also displayed dynamic surface expression dependent on MHC I in contrast to conventional CD8⁺ T cells. Together, these results support a specialized role for CD8α⁺ NK cell populations during Mtb infection correlating with disease resistance.

Fig. 1.. Frequencies of NK cell subpopulations are altered after Mtb infection.

(A) Gating strategy employed for NK cells from a representative sample. NK cells were defined as CD19⁻, CD3⁻, CD14⁻ lymphocytes who expressed any level of either CD16 or CD56. NK cells were further subdivided into three subsets based on CD56 expression. (B) Change in frequency of total NK cells as a proportion of total live population. (C) Frequency of each NK cell subset in clinical groups. Frequency calculated as percent of each subset among total NK cells. Brackets represent Wilcoxon statistical tests, with unadjusted p-values. *p ≤ 0.05, **p ≤ 0.01.

Fig. 2.. NK cells demonstrate loss of CD16 expression during household-associated latent infection.

(A) Frequency of CD16 in CD56^dim NK cells. Brackets represent Wilcoxon tests with unadjusted p-values. Percent positive for 5 activation markers in (B) CD56^bright NK cells, (C) CD56^dim NK cells, and (D) CD56⁻ NK cells. Brackets represent Wilcoxon tests with unadjusted p-values. *p ≤ 0.05, ****p ≤ 0.0001.

Fig. 3.. Mtb resistors demonstrate depressed NK cell function in vitro.

NK cells stimulated overnight by co-culture with K562 cell line compared to plain media alone. (A) Overall results of activation assay for CD56^dim NK cells for four activation markers. Y-axis represents mean percent positive for each activation marker. P values calculated as a global paired Wilcoxon test including all clinical groups. Each clinical group is displayed separately indicated by line color. (B) Percent difference of four activation markers (stimulated minus resting) comparing between clinical groups. Brackets represent Wilcoxon tests with unadjusted p-values. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 4.. CD8α⁺ NK cells are depleted in the blood of cases with household-associated Mtb infection.

(A) Representative gating of CD8α⁺ in CD56^dim NK cells and frequency of CD8α⁺ within CD56^dim NK cells. (B) Representative gating of CD57 in CD56^dim NK cells and boxplots of percent CD57⁺ within CD56^dim NK cells. (C) Expression of select surface markers between CD8α⁺ vs CD8α⁻ CD56^dim NK cells, active TB are excluded. (D) Activation assay with overnight K562 co-culture versus plain media comparing CD8α⁺ vs CD8α⁻ CD56^dim NK cells. Y-values represent percent difference for CD69 and CD107a. Asterisks represent paired Wilcoxon tests. (E) As in panel D, instead displaying CD56⁻ NK cells. P-values represent unadjusted Wilcoxon testing. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 5.. CD8α⁺ NK cells are prevalent in healthy US donors with ubiquitous intracellular CD8α expression.

(A) Flow cytometry plot and percentage of CD8αα⁻, CD8αα⁺, CD8αβ⁺, and CD8ββ⁺ among total NK (N=3). (B) Percentage of CD8α⁺ NK cells in different subsets based on CD56 expression (N=10). (C) Frequency of CD16 expression in CD8α⁺ CD56^dim NK cells. (D) Frequency and representative flow cytometry plots of CD8α expression comparing CD56^dim NK cells and T cells with extracellular (EC), IC (Intracellular), and EC + IC CD8α staining. (E) Percent CD8α⁺ in EC, IC, and EC + IC staining at day 1 and day 7 in CD56^dim NK cells and T cells. Statistical significance was measured using unpaired t tests, ns – no significance, *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

Fig. 6.. CD8α⁺ CD56^dim NK cells have enhanced response to cytokine stimulation.

(A) Pairwise analysis of percent CD8α⁺ NK cells upon stimulation with IL15/12/18 and MtbLys 1:200 (n=9). (B) Pairwise analysis and representative flow plots of intracellular IFNγ in NK CD56^dim upon stimulation with IL15/12/18 and MtbLys at dilution 1:200 (n=9). (C) Percent intracellular IFNγ⁺ in CD8α⁺ versus CD8α⁻ CD56^dim NK cells comparing IL-15-Su, IL15-Su/12/18, IL-12/18, or MtbLys conditions (n=9). Both the CD16⁺ (right) and CD16⁻ (left) CD56^dim NK subpopulations are displayed. (D) Representative flow cytometry plots and paired analysis of percent change CD8α⁺ with anti-MHC I for 15 hours among CD56^dim NK cells (left) and CD8⁺ T cells (right) (n=8). Statistical significance was measured using paired t-test, with threshold of *p<0.05, **p<0.005, ***p<0.001. IFNγ: Interferon γ, Mtb Lys: Mtb whole cell lysate.

Fig. 7.. CITE-seq analysis of CD8α⁺ vs CD8α⁻ NK cells from two healthy donors reveals overlapping transcriptomes.

(A) Results of clustering and uniform manifold approximation (UMAP) projection for all NK cells. (B) UMAP reduction colored by Donor. (C) UMAP reduction colored by condition. (D) Violin plots representing top six genes that were differentially expressed between the four NK cell clusters. (E) Violin plots of the eight differentially expressed genes between resting and Mtb lysate condition. (F) Distribution of NK cell expression of CD8 based on barcoded antibodies against CD8α and CD8A transcript. (G) Violin plot of the expression of CD8A and CD8B transcripts, as well as CD8α surface expression between the four NK cell clusters. (H) Correlation of CD8α expression between transcript and protein measurement. (I) Annotated UMAP based on transcript expression of CD8A into CD8α⁺ and CD8 α⁻ NK cells. (J) Percent of NK cells expressing CD8A transcript between stimulation conditions. (K) Dot-plot of genes differentially expressed between CD8α⁺ and CD8α⁻ NK cells.