Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

doi:10.1128/JVI.02245-16

. 2017 Feb 14;91(5):e02245-16.

doi: 10.1128/JVI.02245-16. Print 2017 Mar 1.

Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

Raphaëlle Riou^{1

2}, Céline Bressollette-Bodin^{3

4

5}, David Boutoille⁶, Katia Gagne^{1

2

7

8}, Audrey Rodallec⁵, Maeva Lefebvre⁶, François Raffi⁶, David Senitzer⁹, Berthe-Marie Imbert-Marcille^{3

4

5}, Christelle Retière^{10

2}

Affiliations

¹ Etablissement Français du Sang, Nantes, France.
² CRCINA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France.
³ Centre de Recherche en Transplantation et Immunologie UMR1064, Université de Nantes, Nantes, France.
⁴ Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France.
⁵ Service de Virologie,CHU Nantes, Nantes, France.
⁶ Service des Maladies Infectieuses et Tropicales, CHU Nantes, Nantes, France.
⁷ Laboratoire d'Histocompatibilité et d'Immunogénétique, EFS Nantes, Nantes, France.
⁸ LabEx Transplantex, Université de Strasbourg, Strasbourg, France.
⁹ Division of Hematology and Bone Marrow Transplantation, City of Hope, National Medical Center, Duarte, California, USA.
¹⁰ Etablissement Français du Sang, Nantes, France christelle.retiere@efs.sante.fr.

PMID: 28031361
PMCID: PMC5309965
DOI: 10.1128/JVI.02245-16

Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

Raphaëlle Riou et al. J Virol. 2017.

. 2017 Feb 14;91(5):e02245-16.

doi: 10.1128/JVI.02245-16. Print 2017 Mar 1.

Authors

Affiliations

¹ Etablissement Français du Sang, Nantes, France.
² CRCINA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France.
³ Centre de Recherche en Transplantation et Immunologie UMR1064, Université de Nantes, Nantes, France.
⁴ Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France.
⁵ Service de Virologie,CHU Nantes, Nantes, France.
⁶ Service des Maladies Infectieuses et Tropicales, CHU Nantes, Nantes, France.
⁷ Laboratoire d'Histocompatibilité et d'Immunogénétique, EFS Nantes, Nantes, France.
⁸ LabEx Transplantex, Université de Strasbourg, Strasbourg, France.
⁹ Division of Hematology and Bone Marrow Transplantation, City of Hope, National Medical Center, Duarte, California, USA.
¹⁰ Etablissement Français du Sang, Nantes, France christelle.retiere@efs.sante.fr.

PMID: 28031361
PMCID: PMC5309965
DOI: 10.1128/JVI.02245-16

Abstract

Primary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C⁺ NK cells, CD16⁺ Vδ2(-) γδ T cells, and conventional HCMV-specific CD8⁺ T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals.IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive immune responses with a deep HCMV imprint. The massive lymphocytosis could be the result of nonadapted or uncontrolled immune responses limiting the effectiveness of the specific responses mounted. Both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage. Furthermore, we cannot exclude a delayed immune response caused by immune escape established by HCMV strains.

Keywords: HCMV; NK cells; T lymphocytes; adaptive immunity; innate immunity.

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Figures

**FIG 1**
Early expansion in PHIP of activated and responsive NK cells that displayed not fully mature NKG2C, NKG2A^hi, KIR2D^lo, and CD57^lo phenotypes. (A) Patterns of cell composition following CD3 and CD56 expression in HCMV^−/+ individuals and PHIP. We summed CD3⁻ CD56⁻, CD3⁻ CD56⁺, CD3⁺ CD56⁺, and CD3⁺ CD56⁻ cell subsets, weighting them according to their frequencies, as indicated. The size of the pie chart is proportional to the absolute number of total lymphocytes. (B) Scatter plots representing the percentages and the absolute numbers (AN) of CD3⁻ CD56⁺ NK cells assessed by flow cytometry in HCMV⁻ (n = 26) or HCMV⁺ (n = 39) individuals and PHIP (n = 17). (C) Representative density plots of CD3⁻ CD56⁺ NK cells expressing KIR2D and NKG2C in HCMV⁻, HCMV⁺ 2C⁺, and HCMV⁺ 2C⁻ individuals and PHIP. (D) Frequencies of total NKG2C⁺, KIR2D⁺, and KIR2D⁺ NKG2C⁺ NK cells for 26 HCMV⁻, 22 HCMV⁺ 2C⁻, and 17 HCMV⁺ 2C⁺ individuals and 16 PHIP. The results are represented as means ± standard errors of the mean (SEM). (E) Frequencies of KIR2DL2/S2/L3⁺ and KIR2DL1/S1⁺ NK cells for 34 HCMV⁻ and 36 HCMV⁺ individuals and 11 PHIP. (F) CD56, CD38, and NKG2D expression (mean fluorescence intensity [MFI]) on NK cells for representative HCMV⁻ and HCMV⁺ individuals and PHIP. The scatter plots represent CD56 (n = 26 HCMV⁻; n = 39 HCMV⁺; n = 17 PHIP), CD38 (n = 26 HCMV⁻; n = 34 HCMV⁺; n = 17 PHIP), and NKG2D (n = 23 HCMV⁻; n = 39 HCMV⁺; n = 16 PHIP) ΔMFI on NK cells from 26 HCMV⁻ and 34 HCMV⁺ individuals and 17 PHIP after deletion of MFI obtained with isotype control (ΔMFI). (G) Scatter plots of KIR2D⁻ NKG2A⁺ and KIR2D⁺ NKG2A⁻ NK frequencies and correlation between the populations observed in all studied groups. (H) Scatter plots of CD57 (n = 26 HCMV⁻; n = 39 HCMV⁺; n = 16 PHIP) and PD-1 (n = 15 HCMV⁻; n = 32 HCMV⁺; n = 14 PHIP) NK cells. (I) Degranulation and IFN-γ secretion capacities of NK cells from 6 HCMV⁻, 14 HCMV⁺, and 5 PHIP PBMCs assessed by flow cytometry after PBMC coculture with medium alone, 721.221 cells for spontaneous-activity measurement, or P815 cells for reverse ADCC measurement. Statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) between more than two groups was determined using one-way ANOVA. (C) Spearman's rank correlation coefficient was calculated when a significant P value was observed (P < 0.05).

**FIG 2**
Early differentiation of CD3⁺ CD56⁻ T cells in PHIP. (A) Frequency (n = 26 HCMV⁻; n = 39 HCMV⁺; n = 17 PHIP) and absolute number (n = 18 HCMV⁻; n = 23 HCMV⁺; n = 13 PHIP) of CD3⁺ CD56⁻ T lymphocytes. (B) Absolute numbers (n = 18 HCMV⁻; n = 23 HCMV⁺; n = 10 PHIP) and frequencies (n = 24/25 HCMV⁻; n = 39 HCMV⁺; n = 10 PHIP) of CD4⁺ and CD8⁺ T cells. Frequencies of PD-1⁺ among CD4⁺ and CD8⁺ T cells (n = 15 HCMV⁻; n = 32 HCMV⁺; n = 14 PHIP). (C) AN and frequencies of NKG2D⁺, DNAM-1⁺, CD57⁺, NKG2A⁺, KIR2DL2/3⁺, and KIR3DL1⁺ T cells. Frequency results are shown as means ± SEM. (D) Density plots illustrating HLA-A2/pp65 pentamer-stained CD3⁺ T lymphocytes from representative HCMV⁺ individuals and PHIP and representative patterns of granzyme A and perforin expression in HLA-A2/pp65-specific cells. The scatter plots represent the absolute number of HLA-A2-pp65-specific CD3⁺ T cells from HCMV⁺ healthy individuals (n = 13) and PHIP (n = 6). (E) Patterns of CD4 and CD8 cell composition following CD27 and CD28 expression for CD8⁺ T cells in HCMV⁺ individuals and PHIP. We summed all cell subsets, weighting them according to their frequencies, as indicated. The size of the pie chart is proportional to the absolute number of total T lymphocytes. (F and G) Density plots illustrating the pattern of CD27/CD28 and CD45RA/CCR7 expression and corresponding scatter plots of frequencies observed in CD8⁺ T cells (F) and HLA-A2–pp65-specific T cells (G) from HCMV⁺ individuals (n = 10) and PHIP (n = 6). Means and SEM are shown. Statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) between more than two groups was determined using one-way ANOVA and between two groups using an unpaired t test.

**FIG 3**
HCMV signature on γδ T cell compartment. (A) Absolute numbers of γδ T cells and frequencies and absolute numbers of Vδ2(−) γδ T cells in 17 HCMV⁻ and 27 HCMV⁺ individuals and 15 PHIP. (B) Representative histogram illustrating γδ T cells stained with Vδ₂ γδ TCR monoclonal antibody. The scatter plots represent CD16⁺ (n = 16 HCMV⁻; n = 26 HCMV⁺; n = 15 PHIP) and CD38⁺ (n = 17 HCMV⁻; n = 26 HCMV⁺; n = 10 PHIP) frequencies observed in Vδ2(+) and Vδ2(−) γδ T cell subsets. (C) Qualitative representation of NKG2C⁺ NK cell frequency as a function of the corresponding Vδ2(−) γδ T cell frequency for each individual for 16 HCMV⁺ 2C⁻, 11 HCMV⁺ 2C⁺, and 13 PHIP. The correlation graphs represent NKG2C⁺ NK cell and NKG2C⁺ T cell frequencies, CD57⁺ NK cell and CD57⁺ T cell frequencies, and CD57⁺ NK cell and PD-1⁺ T cell frequencies in 15 PHIP. (D) Ten-by-10 dot plot representations of Vδ2(−) and Vδ2(+) γδ T cells expressing or not CD16 in all studied groups. Means and SEM are shown. Statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) between more than two groups was determined using one-way ANOVA. (C) Spearman's rank correlation coefficient was calculated when a significant P value was observed (P < 0.05).

**FIG 4**
Phenotypic characterization of CD3⁺ CD56⁺ T cells. (A) Representative histograms illustrating CD38, NKG2A, NKG2C, and CD57 expression on CD3⁺ CD56⁺ T lymphocytes. The scatter plots represent the frequencies of CD38 (n = 26 HCMV⁻; n = 39 HCMV⁺; n = 10 PHIP), NKG2A (n = 26 HCMV⁻; n = 38 HCMV⁺; n = 17 PHIP), NKG2C (n = 25 HCMV⁻; n = 39 HCMV⁺; n = 14 PHIP), and CD57 (n = 26 HCMV⁻; n = 39 HCMV⁺; n = 16 PHIP) CD3⁺ CD56⁺ T lymphocytes. (B) Representative density plots illustrating CD161 and ILT-2 expression on CD56⁺ T lymphocytes (n = 26 HCMV⁻; n = 37 HCMV⁺; n = 11 PHIP). The scatter plots represent the frequencies of KIR2D, CD161, and ILT-2 on CD56⁺ T lymphocytes in HCMV⁻ and HCMV⁺ individuals and PHIP. Means and SEM are shown. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

**FIG 5**
Dynamics of NK, γδ, and T cell subsets following primary HCMV infection. (A) Density plots illustrating all lymphocyte frequencies using CD3/CD56 marker combinations at different time points: acute infection (P20), month 4 (M4) (P20), and month 9 (M9) (P19). The histograms represent CD38 expression on NK cells (CD3⁻ CD56⁺) at all 3 kinetic points (acute, M4, and M9), and the density plots illustrate KIR2D⁺, NKG2C⁺, CD57⁺, and NKG2D⁺ NK cells. (B) Correlation graphs representing NKG2A⁺ NK cell; CD57⁺ NK cell; and CD38⁺, NKG2D⁺, PD-1⁺, and CD45RA⁺ T cell frequencies and postinfection days for 17 PHIP. Spearman's rank correlation coefficient was calculated when a significant P value was observed (P < 0.05). (C) Density plots representing CD8⁺ and CD45RA⁺ αβ T lymphocytes at all 3 kinetic points (acute, M4, and M9). The histograms represent Vδ2(−) T lymphocytes and CD16⁺ γδ T cells among Vδ2(−) T lymphocytes at all 3 kinetic points.

**FIG 6**
Profound genetic modulation induced by HCMV in PHIP. (A) PCA illustrating genetic-profile variability in two-dimensional scale after quality control and data normalization. Each dot in the plot represents a studied individual (HCMV⁻, n = 13; HCMV⁺ 2C⁻, n = 12; HCMV⁺ 2C⁺, n = 12; and PHIP, n = 11). (B) Heat maps generated after hierarchical clustering using Cluster 3.0 and Treeview for HCMV⁻ (n = 13) versus PHIP (n = 11) and HCMV⁺ 2C⁺ (n = 12) versus PHIP data set comparisons. Downregulated clusters in PHIP are shown in red, and upregulated clusters in PHIP are indicated in green. The bars indicate the numbers of GO categories belonging to the immune response, cell cycle, and other cell functions in each cluster for each data set comparison after submission to GOminer software to assess biological significance. GO categories were selected with a cutoff of enrichment of >1.5, an FDR of <0.01, and >5 changed genes. (C) FCs for all upregulated genes with an FC of >3 involved in immune response in PHIP compared to an HCMV⁻ healthy individual gene expression profile (FC of >10, dark green; FC of >4, green; 3 < FC > 4, light green). (D) FCs for DEG between HCMV⁻ healthy individuals and PHIP for whom protein expression was assessed by flow cytometry. Upregulated and downregulated genes are indicated in green and red, respectively. The P value observed for each gene is indicated.

**FIG 7**
Schematic representation of main phenotypic modulations on T cell (CD56⁻, CD56⁺, and γδ) and NK cell compartments. Inhibitory receptors are indicated in red, activating receptors in green, receptors with increased MFI in blue, and markers associated with activated status in orange. The increased ratio of absolute lymphocyte counts in PHIP is indicated at the bottom left.

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References

1. van de Berg PJEJ, van Stijn A, Ten Berge IJM, van Lier RAW. 2008. A fingerprint left by cytomegalovirus infection in the human T cell compartment. J Clin Virol 41:213–217. doi:10.1016/j.jcv.2007.10.016. - DOI - PubMed
1. Sissons JGP, Wills MR. 2015. How understanding immunology contributes to managing CMV disease in immunosuppressed patients: now and in future. Med Microbiol Immunol 204:307–316. doi:10.1007/s00430-015-0415-0. - DOI - PubMed
1. Biron CA, Byron KS, Sullivan JL. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N Engl J Med 320:1731–1735. doi:10.1056/NEJM198906293202605. - DOI - PubMed
1. Orange JS. 2002. Human natural killer cell deficiencies and susceptibility to infection. Microbes Infect 4:1545–1558. doi:10.1016/S1286-4579(02)00038-2. - DOI - PubMed
1. Saunders PM, Vivian JP, O'Connor GM, Sullivan LC, Pymm P, Rossjohn J, Brooks AG. 2015. A bird's eye view of NK cell receptor interactions with their MHC class I ligands. Immunol Rev 267:148–166. doi:10.1111/imr.12319. - DOI - PubMed

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[1] van de Berg PJEJ, van Stijn A, Ten Berge IJM, van Lier RAW. 2008. A fingerprint left by cytomegalovirus infection in the human T cell compartment. J Clin Virol 41:213–217. doi:10.1016/j.jcv.2007.10.016. - DOI - PubMed

[2] van de Berg PJEJ, van Stijn A, Ten Berge IJM, van Lier RAW. 2008. A fingerprint left by cytomegalovirus infection in the human T cell compartment. J Clin Virol 41:213–217. doi:10.1016/j.jcv.2007.10.016. - DOI - PubMed

[3] Sissons JGP, Wills MR. 2015. How understanding immunology contributes to managing CMV disease in immunosuppressed patients: now and in future. Med Microbiol Immunol 204:307–316. doi:10.1007/s00430-015-0415-0. - DOI - PubMed

[4] Sissons JGP, Wills MR. 2015. How understanding immunology contributes to managing CMV disease in immunosuppressed patients: now and in future. Med Microbiol Immunol 204:307–316. doi:10.1007/s00430-015-0415-0. - DOI - PubMed

[5] Biron CA, Byron KS, Sullivan JL. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N Engl J Med 320:1731–1735. doi:10.1056/NEJM198906293202605. - DOI - PubMed

[6] Biron CA, Byron KS, Sullivan JL. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N Engl J Med 320:1731–1735. doi:10.1056/NEJM198906293202605. - DOI - PubMed

[7] Orange JS. 2002. Human natural killer cell deficiencies and susceptibility to infection. Microbes Infect 4:1545–1558. doi:10.1016/S1286-4579(02)00038-2. - DOI - PubMed

[8] Orange JS. 2002. Human natural killer cell deficiencies and susceptibility to infection. Microbes Infect 4:1545–1558. doi:10.1016/S1286-4579(02)00038-2. - DOI - PubMed

[9] Saunders PM, Vivian JP, O'Connor GM, Sullivan LC, Pymm P, Rossjohn J, Brooks AG. 2015. A bird's eye view of NK cell receptor interactions with their MHC class I ligands. Immunol Rev 267:148–166. doi:10.1111/imr.12319. - DOI - PubMed

[10] Saunders PM, Vivian JP, O'Connor GM, Sullivan LC, Pymm P, Rossjohn J, Brooks AG. 2015. A bird's eye view of NK cell receptor interactions with their MHC class I ligands. Immunol Rev 267:148–166. doi:10.1111/imr.12319. - DOI - PubMed

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Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

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Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

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