In Utero Activation of Natural Killer Cells in Congenital Cytomegalovirus Infection

Abstract

Background: Congenital cytomegalovirus (CMV) infection is the most common infectious cause of birth defects and neurological damage in newborns. Despite a well-established role for natural killer (NK) cells in control of CMV infection in older children and adults, it remains unknown whether fetal NK cells can sense and respond to CMV infection acquired in utero.

Methods: Here, we investigate the impact of congenital CMV infection on the neonatal NK-cell repertoire by assessing the frequency, phenotype, and functional profile of NK cells in cord blood samples from newborns with congenital CMV and from uninfected controls enrolled in a birth cohort of Ugandan mothers and infants.

Results: We find that neonatal NK cells from congenitally CMV infected newborns show increased expression of cytotoxic mediators, signs of maturation and activation, and an expansion of mature CD56- NK cells, an NK-cell subset associated with chronic viral infections in adults. Activation was particularly prominent in NK cell subsets expressing the Fcγ receptor CD16, indicating a role for antibody-mediated immunity against CMV in utero.

Conclusions: These findings demonstrate that NK cells can be activated in utero and suggest that NK cells may be an important component of the fetal and infant immune response against CMV.

Clinical trials registration: NCT02793622.

Keywords: CD56-negative NK cells; NK cells; NKG2C; congenital CMV; cord blood; cytomegalovirus; flow cytometry; neonatal immunity.

Figure 1

CD56^neg NK cells expand in newborns with congenital CMV infection. A, Gating strategy for total NK cells and NK-cell subsets. NK cells are defined as lymphocytes > single cells > CD3⁻/CD14⁻/CD19⁻/CD7⁺ > CD56⁺ and/or CD16⁺. NK subsets are defined based on their relative expression of CD56 and CD16. Total NK cells is the sum of the 3 gated subsets. B, Frequency of total NK cells in CMV-infected (cCMV-positive, n = 16) and uninfected (cCMV-negative, n = 69) newborns. C, Frequency of NK-cell subsets out of the total NK cells in cord blood derived from cCMV-positive (n = 16) and cCMV-negative (n = 69) newborns. D, Density of CD7 expression on NK cells measured as normalized MFI (median) between cCMV-positive (n = 16) and cCMV-negative (n = 69) infants. *P < .05, ***P < .001, Wilcoxon rank sum test. Abbreviations: SSC-A, side scatter area; FSC-A, forward scatter area; cCMV, congenital CMV; CMV, cytomegalovirus; MFI, mean fluorescence intensity; NK cell, natural killer cell.

Figure 2

NK cells from cCMV-positive neonates express higher levels of cytotoxic mediators. A, Frequency of cord blood NK cells expressing granzyme B, perforin, and granulysin from cCMV-positive newborns (n = 16) and cCMV-negative controls (n = 69). Granulysin expression was evaluated on a smaller subset of cord blood samples (cCMV-positive, n = 8; cCMV-negative, n = 36). NK cells are defined as lymphocytes > single cells >CD3⁻/CD14⁻/CD19⁻ > CD56⁺ and/or CD16⁺. Note that CD7 was not included in the flow cytometry panel used to evaluate markers listed in Figure 2. See details in Supplementary Figure 2B. B, Density of granzyme B, perforin, and granulysin expression on NK-cell subsets measured as normalized MFI from cCMV-positive newborns (n = 16) and cCMV-negative controls (n = 69). C, Proportion of NK-cell subsets expressing the listed combination of granzyme B and perforin in cCMV-negative (n = 69) and cCMV-positive infants (n = 16). Coexpression was calculated using Boolean gating in FlowJo and pie graphs, depicting average proportions, were generated in SPICE. D, Frequency of NK cells expressing T-bet, eomesodermin, and Ki67 in cCMV-positive infants (n = 16) and cCMV-negative controls (n = 69). *P < .05, **P < .01, ***P < .001, Wilcoxon rank sum test. Abbreviations: cCMV, congenital cytomegalovirus; GzB, granzyme B; MFI, mean fluorescence intensity; NK cell, natural killer cell; Pfn, perforin.

Figure 3

NK cells show altered expression of NKRs in cCMV-positive newborns. Frequency of NK cells expressing NKG2A, NKp30, NKG2C, CD57, and LILRB1 in cord blood derived from cCMV-positive (n = 16) and cCMV-negative (n = 69) newborns. NK cells are defined as lymphocytes > single cells > CD3⁻/CD14⁻/CD19⁻/CD7⁺ > CD56⁺ and/or CD16⁺. For NK-cell gating strategy refer to Figure 1A. Individuals with < 2% of their total NK cells expressing NKG2C⁺ are highlighted in red/below dotted line in the NKG2C panel. Axes for % CD57⁺ and % LILRB1⁺ range from 0% to 50%. *P < .05, **P < .01, Wilcoxon rank sum test. Abbreviations: cCMV, congenital cytomegalovirus; NK cell, natural killer cell; NKR, NK receptor.

Figure 4

NK-cell phenotype in symptomatic and asymptomatic cCMV cases. A, Frequency of total NK cells defined as CD3⁻/CD14⁻/CD19⁻/CD7⁺ lymphocytes in cord blood from symptomatic (n = 5, dark green) and asymptomatic (n = 11) cCMV-positive newborns. Newborns are defined as symptomatic if they demonstrated severe microcephaly and/or were small for gestational age at birth. B, Frequency of NK-cell subsets out of total NK cells in cord blood derived from symptomatic (n = 5) and asymptomatic (n = 11, light green) cCMV-positive newborns. C, Frequency of NK cells expressing granzyme B, perforin, granulysin, T-bet, eomesodermin, and Ki67 in symptomatic (n = 5) and asymptomatic (n = 11) cCMV-positive newborns. D, Frequency of NK cells expressing NKG2A, NKp30, NKG2C, CD57, and LILRB1 in symptomatic (n = 5) and asymptomatic (n = 11) cCMV-positive newborns. **P < .01. For gating strategy of NK-cell subsets refer to Figure 1A for (A, B, and D), and Supplementary Figure 2B for (C). Abbreviations: cCMV, congenital cytomegalovirus; NK cell, natural killer cell.