Expansion of a unique CD57⁺NKG2Chi natural killer cell subset during acute human cytomegalovirus infection

Abstract

During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94-NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56(dim)CD16(+) NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94-NKG2C receptor and CD57 in CMV(+) donors. These CD57(+)NKG2C(hi) NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57(+)NKG2C(hi) NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57(+)NKG2C(hi) NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C(+) NK cells proliferated, became NKG2C(hi), and finally acquired CD57. Thus, we propose that CD57 might provide a marker of "memory" NK cells that have been expanded in response to infection.

Fig. 1.

A unique population of CD57⁺NKG2C^hi NK cells is detected in CMV⁺ donors. (A) CD3^negCD56^dimCD16⁺ NK cells from four CMV⁻ (Upper) and CMV⁺ (Lower) donors were gated and analyzed for expression of CD57 and NKG2C. (B) The geometric mean fluorescence intensity (gMFI) for NKG2C in the CD57⁺ NK cell subset from CMV⁻ and CMV⁺ donors is shown. (C) The percentage of CD57⁺NKG2C^hi cells in the CD56^dimCD16⁺ NK cell subset from CMV⁺ and CMV⁻ donors is shown. (D) The ratio of CD57⁺NKG2C^hi/CD57⁻NKG2C^hi NK cells for each CMV⁻ and CMV⁺ donors is shown. Triangles represent CMV⁻ donors (n = 23), circles represent CMV⁺ donors (i.e., seropositive and/or CMV-specific T-cell responses; n = 29).

Fig. 2.

CD57⁺NKG2C^hi NK cells from CMV⁺ donors degranulated when activated through NKG2C. PBMCs from CMV⁺ donors where stimulated with plate-bound antibodies against CD56 (negative control), NKG2C, or CD16 (positive control). The percentage of CD107α⁺ cells was determined for CD57⁺NKG2C^hi NK cells (n = 7).

Fig. 3.

Certain inhibitory NK receptors are underrepresented on the CD57⁺NKG2C^hi NK cell subset from CMV⁺ donors. (A) CD56^dimCD16⁺ NK cells from CMV⁻ (Left) and CMV⁺ (Right) donors were gated and analyzed for expression of CD57 and NKG2C. CD57⁺NKG2C⁻ (a), CD57⁺NKG2C^dim (b), and CD57⁺NKG2C^hi (c) NK cells were assessed for CD56 and NKG2A expression. (B) The percentage of NKG2A⁺ cells was determined on CD57⁺NKG2C⁻ and CD57⁺NKG2C^hi NK cells from CMV⁺ donors (n = 16). (C) The percentage of KIR3DL1⁺ cells was determined on CD57⁺NKG2C⁻ and CD57⁺NKG2C^hi NK cells from CMV⁺ donors. Graphs from HLA-Bw4⁺ (Upper; n = 9) and HLA-Bw4⁻ donors (Lower; n = 6) are shown. (D) The percentage of KIR2DL1⁺ cells was determined on CD57⁺NKG2C⁻ and CD57⁺NKG2C^hi NK cells for CMV⁺ donors who have the KIR2DL1 ligand (Left; n = 5) or do not (Right; n = 7).

Fig. 4.

CD57⁺NKG2C^hi NK cells increase during acute CMV infection in SOT recipients. (A) NKG2C and CD57 expression on CD56^dimCD16⁺ NK cells from SOT recipients with acute CMV infection was analyzed. Antiviral treatment was administrated immediately after detection of viremia (day 0). The percentage of CD57⁺NKG2C^hi NK cells detected over time and the CMV viral load detected in plasma (value > 400 copies/mL) from representative patients are shown (n = 6). Solid line and square represent CD57⁺NKG2C^hi NK cells, dashed line and triangle represent CMV DNA. (B and C) The percentages of CD57⁺NKG2C^hi NK cells detected over time from one representative SOT recipient without CMV viremia (B; n = 5) and from a healthy CMV⁺ donor (C) are shown. (D and E) Approximate absolute numbers (in 10³ cells/L) of CD57⁺NKG2C^hi NK cells detected over time in one representative SOT recipient with CMV viremia (D; CMV viral load shown; n = 6) and in SOT recipients without CMV viremia (E; n = 5).

Fig. 5.

During CMV acute infection, NKG2C⁺ NK cells acquire CD57 expression and become CD57⁺NKG2C^hi NK cells. (A and B) The percentages of CD57⁺ NK cells detected over time in SOT recipients with acute CMV infection (A; n = 6) or without CMV viremia (B; n = 5) are shown. (C) CD56^dimCD16⁺ NK cells were analyzed for expression of CD57 and NKG2C. Plots for different time points from a representative SOT recipient with CMV viremia are shown (n = 6). Population gates: CD57⁻NKG2C^dim, CD57⁻NKG2C^hi, CD57^dimNKG2C^hi, and CD57⁺NKG2C^hi NK cells. (D) CD56^dimCD16⁺ NK cells were gated on NKG2C⁺ and NKG2C⁻ cells and the percentage of CD57⁺ cells for each subset was assessed over time. The ratio of CD57⁺/CD57⁻ cells on each subset from a representative SOT recipient with CMV viremia is shown (n = 6). Solid square is from NKG2C⁺, solid triangle is from NKG2C⁻ NK cells.

Fig. 6.

All NK cells divide in response to acute CMV infection. (A and B) CD56^dimCD16⁺ NK cells were gated on NKG2C⁺ and NKG2C⁻ cells, and Ki-67 expression on all CD56^dimCD16⁺ NK cells and in each subset was assessed. Graphs from representative SOT recipients with CMV viremia (A; n = 4) and without CMV viremia (B) are shown. Solid line and empty circle represent CD56^dimCD16⁺ NK cells, solid line and square represent NKG2C⁺ NK cells, and dashed line and triangle represent NKG2C⁻ NK cells. (C) The percentage of Ki-67⁺ cells within the CD56^dimCD16⁺ NK cells detected over time in healthy CMV⁺ donors is shown.