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Dichotomous effects of latent CMV infection on the phenotype and functional properties of CD8+ T-cells and NK-cells
Cellular Immunology 300 (2016) 26–32
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Research paper
Dichotomous effects of latent CMV infection on the phenotype and functional properties of CD8+ T-cells and NK-cells Austin B. Bigley a, Guillaume Spielmann a,b, Nadia Agha a, Daniel P. O’Connor a, Richard J. Simpson a,⇑ a b
Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, TX 77204, USA School of Kinesiology, Louisiana State University, 112 Long Fieldhouse, Baton Rouge, LA 70803, USA
a r t i c l e
i n f o
Article history: Received 9 October 2015 Revised 9 November 2015 Accepted 23 November 2015 Available online 24 November 2015 Keywords: NKG2C CD57 CD27 CD28 Cytotoxicity NKCA K562 pp65 IE1 Immunosenescence Aging Viral load
a b s t r a c t CMV markedly alters the phenotype and function of NK-cells and T-cells and has been linked to immunosenescence. We show here that subjects with effective CMV control (evidenced by low CMV IgG titers) have functional responses to CMV that are driven by either NKG2C+ NK-cells or CMVspecific T-cells (15 of 24 subjects), but not both. These data indicate that people with effective CMV control are either NK-cell or T-cell responders, and corroborates the idea that NK-cells have rheostat-like properties that regulate anti-viral T-cell responses. Whether or not lifelong CMV control through either NK-cell or T-cell responses have implications for immunosenescence remains to be determined. Ó 2016 Published by Elsevier Inc.
1. Introduction CMV is a prevalent b-herpesvirus that infects 50–80% of all adults in the United States [1]. Within the T-cell compartment, CMV is often considered to be an immunological burden that exerts mostly negative effects on immune status and overall health [2]. This has led to the perception that it may be beneficial to eradicate CMV or vaccinate against it as a matter of public health [3]. However, recent evidence suggests that latent herpesviruses (including CMV) may play a pivotal role in cancer immunosurveillance by ‘arming’ NK-cells to adequately destroy malignant target cells [4,5]. For example, NK-cells in mice with latent Murid herpesvirus 4 infection show increased Granzyme B expression, IFN-c production and cytotoxicity, which protects infected mice from a lethal lymphoma challenge [4]. Further, we have reported increased NK-cell-mediated cytotoxicity against HLA-E+ human tumor cell lines in healthy people previously exposed to CMV [5]. Thus, it may be that CMV exerts divergent effects on NK-cell and
⇑ Corresponding author. E-mail address: [email protected] (R.J. Simpson). http://dx.doi.org/10.1016/j.cellimm.2015.11.005 0008-8749/Ó 2016 Published by Elsevier Inc.
T-cell immunity, with beneficial effects being found primarily within the NK-cell compartment. The functional effects of latent CMV infection on NK-cells and Tcells have been linked to clonal expansion of ‘‘CMV-specific” NKcell and T-cell subsets [6]. Within the NK-cell compartment, expansion of NK-cells expressing the activating receptor NKG2C is associated with greater viral control [7] due to increased lysis of CMV-infected cells that express HLA-E, the ligand for NKG2C [8]. This clonal expansion is associated with increased differentiation, which can result in functional exhaustion and replicative senescence, particularly in the T-cell compartment [9]. Highlydifferentiated T-cells can be defined by loss of CD28 and CD27 expression and NK-cell differentiation can be defined by acquisition of the terminal differentiation marker CD57 [10]. It has long been known that CMV is able to drive the preferential expansion of highly-differentiated NKG2C+ NK-cells [11] and CD8+ T-cells [9]. These CMV-driven responses are highly variable between CMV-infected subjects [12] and the differentiation status of Tcells has been reported to be independent of NK-cell differentiation in those with CMV [13]. It remains to be seen, however, how CMVspecific T-cell responses correlate with those of NK-cells at a func-
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tional level. If CMV-specific NK-cell and T-cell functional responses are reciprocally related, it may be that CMV boosts immunity in some, while inhibiting it in others. It is essential, therefore, that we understand the different mechanisms by which CMV is controlled in the healthy host before we consider eradication of CMV, as such an approach may have unforeseen consequences for anti-viral and anti-tumor immunity. The primary aims of this study were twofold. First, we measured NK-cell and T-cell differentiation in CMVpos subjects and defined ‘CMV response’ categories based on the relative magnitude of NK-cell and T-cell differentiation. Second, we determined the effect of ‘CMV response’ category on NK-cell cytotoxicity, CMVspecific functional T-cell responses, and CMV IgG antibody titers. We showed for the first time that CMV-specific NK-cell and T-cell responses are inversely related in healthy subjects with effective CMV control. 2. Methods 2.1. Subjects Forty-eight healthy adults (50% CMVpos; age: 32.3 ± 4.6 yr) voluntarily participated in this study. Subjects were between the ages of 18 and 50 and not taking any immunomodulatory medication. Abstinence from alcohol, caffeine and vigorous physical activity 24 h prior to blood donation, as well as elimination of vitamin/ mineral supplementation at least 4 weeks prior to taking part in the study was required and confirmed verbally with the subjects on their arrival to the laboratory. All subjects provided written informed consent prior to participating in the study and the Committee for the Protection of Human Subjects at the University of Houston approved the protocol.
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controls, respectively. The PepMixes for each viral antigen were assessed in separate assays. Spot-forming cells were enumerated by Zellnet Consulting Inc. (Fort Lee, NJ, USA) and compared with input cell numbers to obtain the frequency of virus-reactive Tcells. The number of spot forming cells in each assay was adjusted for the total CD3+ T-cell count.
2.4. NK-cell cytotoxicity assay As we have described previously [14], monocyte-depleted lymphocytes were co-cultured with anti-CD71 (IgG1, Clone OKT-9) FITC-labeled K562 cells (1.0 105 cells) at 1:1, 2.5:1, 5:1, and 10:1 lymphocyte: K562 cell ratios in a final volume of 2.2 mL of 10% FBS-RPMI 1640. After 4 h incubation at 37 °C, the cells were washed and stained with PE-conjugated anti-CD3 (IgG1, Clone UCHT1) and APC-conjugated anti-CD56 (IgG1, Clone CMSSB) antibodies to quantify the number of NK-cells in each tube. After a final wash, propidium iodide (PI) was added and the number of NKcells, live target cells, and dead target cells was resolved using 4color flow cytometry [16]. All antibodies and the PI were purchased from eBioscience. The K562 cells were purchased from ATCC (Manassas, VA, USA).
2.5. CMV serology Fasting serum samples were frozen at 80 °C until measurement of CMV IgG antibodies, which were analyzed in duplicate using commercially available ELISA kits (BioCheck, Foster City, CA, USA) and a 96-well microplate reader (Molecular Devices, Sunnyvale, CA, USA). Antibody titers were calculated by comparing serum samples to references of known concentrations in accordance with the manufacturer’s instructions.
2.2. Flow cytometry Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Histopaque per the manufacturer’s instructions (Sigma–Aldrich, St. Louis, MO, USA). Aliquots of 1 106 isolated cells were incubated for 30 min with 50 lL of pre-diluted APC-conjugated anti-CD3 (IgG2a, Clone UCHT1), PECy5-conjugated anti-CD8 (IgG2a, Clone MEM-31) or PerCPeFluor710-conjugated anti-CD56 (IgG1, Clone CMSSB) or antiCD4 (IgG1, Clone SK3), Alexa488-conjugated anti-NKG2C (IgG1, Clone 134591) or anti-CD28 (IgG1, Clone CD28.2), and PEconjugated anti-CD57 (IgM, Clone TB01) or anti-CD27 (IgG1, Clone O323) monoclonal antibodies. The anti-CD56, anti-CD8, anti-CD4, anti-CD3, anti-CD57, anti-CD28, and anti-CD27 antibodies were purchased from eBioscience (San Diego, CA, USA) and the antiNKG2C antibody was purchased from R&D Systems (Minneapolis, MN, USA). Lymphocyte phenotype and cell count were assessed by 4-color flow cytometry using an Accuri C6 flow cytometer (Accuri, Ann Arbor, MI, USA) as previously described [14]. 2.3. Frequency and epitope specificity of CMV specific T-cells Enzyme-linked immunospot (ELISPOT) analysis was used to determine the frequency of T-cells secreting IFN-c in response to CMVpp65 or CMV IE-1 PepMixes (JPT Peptide Technologies, Berlin, Germany), which contain all 15mer peptides of each viral antigen in one pool [15]. T-cell responses to antigens of Eptein-Barr Virus (EBV) [Imp-2 and BMLF-1] and Varicella Zoster Virus (VZV) [IE62-A, IE62-B, and IE63] were also measured. ELISPOT assays were performed on PBMCs isolated in CMVpos donors. PBMCs that were unstimulated or stimulated with 1 lg/mL PHA (Sigma– Aldrich, St. Louis, MO, USA) served as negative and positive
2.6. Statistical analysis Data was statistically analyzed using the Predictive Analytics SoftWare (PASW 22.0) statistics computer program. CMVpos subjects (N = 24) were stratified based on their NK-cell and CD8+ Tcell differentiation responses. A high NK-cell differentiation response (NKhigh) was defined as an NKG2C+/CD57+ NK-cell proportion greater than the upper bound of the 95% confidence interval for CMVneg subjects, which was 13% of total NK-cells. A high Tcell differentiation response (Thigh) was defined as a CD28 /CD27 CD8+ T-cell proportion greater than the upper bound of the 95% confidence interval for CMVneg subjects, which was 13% of total CD8+ T-cells. Individuals with a proportion of differentiated NKcells and CD8+ T-cells less than the upper bound of the 95% confidence interval for CMVneg subjects (i.e. <13%) were defined as NKlow and Tlow, respectively. This allowed us to separate CMVpos subjects into 4 discrete response categories: (1) NKhigh/Tlow, (2) NKlow/Thigh, (3) NKhigh/Thigh, and (4) NKlow/Tlow. The effects of CMV serostatus and response category on the proportion and number of lymphocyte subsets were determined using independent sample t-tests and one-way ANOVA, respectively. To examine the effects of CMV serostatus and response category on NK-cell cytotoxicity, a maximum likelihood linear mixed model (LMM) was used that included main effects for dose (1X, 2.5X, 5X, or 10X) and CMV serostatus or ‘CMV response’ category as well as interaction effects of CMV serostatus x dose and ‘CMV response’ category x dose. Bonferroni post hoc analysis was performed to determine the locations of the significant effects for ‘CMV response’ category and dose. The upper bound of the 95% confidence interval was defined as the mean + (1.96 * standard deviation). Statistical significance was defined as p < 0.05.
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3. Results 3.1. Divergence of NK-cell and T-cell differentiation in response to latent CMV infection The effects of latent CMV infection and ‘CMV response’ category on the proportions of highly differentiated NKG2C + NK-cells and CD8+ T-cells are described in Fig. 1A. The proportions of NKG2C +/CD57 + NK-cells and CD28 /CD27 CD8+ T-cells were greater in CMVpos subjects (p < 0.001). Within the CMVpos cohort (N = 24), 6 were NKhigh/Tlow, 9 were NKlow/Thigh, 5 were NKhigh/Thigh, and 4 were NKlow/Tlow. There was no difference in age between these ‘CMV response’ categories (p > 0.05). Representative flow cytometry dot-plots that illustrate the divergence of NK-cell and T-cell differentiation during latent CMV infection are shown in Fig. 1B. The proportion of NKG2C+/CD57+ NK-cells was elevated in the NKhigh/ Tlow and NKhigh/Thigh groups (p < 0.001). The proportion of CD28 /
CD27 /CD8+ T-cells was increased and the proportion of ‘‘naïve” CD28+/CD27+/CD8+ T-cells was decreased in the NKlow/Thigh and NKhigh/Thigh groups (p < 0.001). Additionally, the proportion of CD28 /CD27 /CD4+ T-cells was increased in CMVpos subjects (p < 0.001), but there were no differences based on ‘CMV response’ category (p > 0.05). The individual NK-cell and T-cell differentiation responses of the 24 CMVpos subjects are shown in Fig. 1C. The effects of latent CMV infection and ‘CMV response’ category on individual NK-cell and T-cell subsets and CMV IgG antibody titers are presented in Table 1. The numbers of NKG2C+/CD57+ and NKG2C+/CD57 NK-cells were greater in the NKhigh/Tlow and NKhigh /Thigh groups (p < 0.001); the number of CD28 /CD27 CD8+ T-cells was greater in the NKlow/Thigh and NKhigh/Thigh groups (p < 0.001); the numbers of CD28+/CD27+ CD4+ and CD8+ T-cells, and total CD4+ T-cells were greater in the NKlow Tlow group (p < 0.05); and the number of total CD8+ T-cells was lower in the NKhigh/Tlow group (p < 0.01). CMV IgG antibody titers are elevated
Fig. 1. The CMV-driven accumulation of NKG2C+/CD57+ NK-cells is inversely related to that of CD8+/CD28 /CD27 T-cells in a majority of subjects. (a) Shows the proportions of NKG2C+/CD57 + NK-cells (% of total NK-cells) and CD8+/CD28 /CD27 T-cells (% of total CD8+ T-cells) based on CMV serostatus and ‘CMV response’ category. Values are mean ± SE. A statistically greater proportion of NKG2C+/CD57 + NK-cells or CD8+/CD28 /CD27 T-cells based on CMV serostatus (CMVpos > CMVneg) or CMV response category (e.g. NKhigh > NKlow, Thigh > Tlow) is indicated by #p < 0.001. (b) Displays representative flow cytometry dot-plots for co-expression of NKG2C with CD57 on NK-cells and CD28 with CD27 on CD8+ T-cells. (c) Presents the interaction between NK-cell and T-cell differentiation in individual CMVpos subjects (N = 24). The gray dashed line represents the upper bound of the 95% confidence interval for the CMVneg cohort (N = 24).
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Table 1 Lymphocyte subset numbers contrasted by CMV serostatus and CMV response category. Main effects among the nominal variables (CMV serostatus and CMV response category) are shown with significance indicated by *(p < 0.05). Statistical differences between CMV response categories are indicated by #(p < 0.05). Data are mean ± SD. CMVpos NKhigh Tlow [N = 6]
CMVpos NKlow Thigh [N = 9]
CMVpos NKhigh Thigh [N = 5]
CMV serostatus
CMV response category
NK-cells NKG2C+/CD57+ NKG2C+/CD57 NKG2C-/CD57+ NKG2C-/CD57
246.0 ± 77.9 96.7 ± 41.9# 30.8 ± 19.1# 58.4 ± 70.2 60.1 ± 24.3
201.0 ± 80.9 8.2 ± 6.5 6.2 ± 4.4 106.4 ± 58.3 80.2 ± 28.4
216.2 ± 54.6 66.0 ± 44.1# 22.5 ± 9.5# 72.7 ± 12.8 55.0 ± 20.7
194.4 ± 55.2 4.8 ± 1.6 8.2 ± 4.2 90.8 ± 63.1 90.6 ± 33.7
167.1 ± 80.0 5.7 ± 4.9 8.9 ± 7.3 67.8 ± 53.5 84.7 ± 46.4
3.8 (0.06) 13 (<.001)⁄ 4.8 (0.03)⁄ 2.6 (0.11) 1.8 (0.19)
0.6 (0.61) 17 (<.001)⁄ 7 (<.01)⁄ 0.3 (0.81) 2.5 (0.89)
CD8+ T-cells CD28+/CD27+ CD28+/CD27 CD28 /CD27+ CD28 /CD27
189.0 ± 15.8# 152.3 ± 16.5 10.0 ± 6.5 16.3 ± 8.1 10.3 ± 1.5
369.8 ± 105.7 228.3 ± 34.7 13.7 ± 12.2 64.7 ± 50.0 90.4 ± 34.6#
352.7 ± 98.3 186.0 ± 43.3 12.0 ± 9.6 50.5 ± 26.9 104.2 ± 36.7#
417.8 ± 88.4 328.5 ± 23.3# 6.8 ± 6.9 61.7 ± 71.6 20.8 ± 9.5
342.6 ± 161.4 285.4 ± 154.6 8.9 ± 7.3 34.2 ± 22.3 14.1 ± 11.6
0.2 (0.63) 1.5 (0.23) 0.8 (0.39) 1.5 (0.23) 17 (<.001)⁄
5.1 (<.01)⁄ 23 (<.001)⁄ 0.6 (0.63) 0.7 (0.54) 17 (<.001)⁄
CD4+ T-cells CD28+/CD27+ CD28+/CD27 CD28 /CD27+ CD28 /CD27
541.0 ± 115.6 508.3 ± 101.2 22.7 ± 2.8 2.3 ± 1.5 7.7 ± 12.4
681.3 ± 203.5 623.7 ± 212.6 41.5 ± 25.4 1.3 ± 1.0 14.8 ± 16.8
654.5 ± 181.3 574.0 ± 137.8 57.0 ± 28.7 2.8 ± 2.2 20.7 ± 7.0
1282.8 ± 376.5# 1172.8 ± 357.0# 74.8 ± 47.6 1.5 ± 1.2 33.7 ± 32.0
968.1 ± 381.2 923.1 ± 364.9 39.3 ± 19.8 4.8 ± 6.1 0.9 ± 1.6
1.1 2.3 2.8 0.1 7.5
7.2 7.9 1.7 0.4 1.6
2.6 ± 2.0
4.3 ± 2.1
17.2 ± 7.8#
16.0 ± 6.8#
0.3 ± 0.2
Cell count (cells/ll)
CMV IgG (IU/mL)
in subjects in the NKhigh/Thigh and NKlow/Tlow groups relative to those in the NKhigh/Tlow and NKlow/Thigh groups (p < 0.01). 3.2. CMV-specific T-cell responses are uncoupled from CMV-driven accumulation of highly-differentiated NKG2C+ NK-cells and NK-cellmediated cytotoxicity The effect of ‘CMV response’ category on the number of CMVspecific T-cells is described in Fig. 2A. The number of T-cells responding to the CMV pp65 antigen was greater in the NKlow/Thigh and NKhigh/Thigh groups (p < 0.01) and the number of T-cells responding to the CMV IE-1 antigen was greater in the NKlow/Thigh group (p < 0.05). All of the CMVpos subjects had viral-responsive cells for EBV and VZV, but there was no effect of ‘CMV response’ category on the number of EBV Imp-2 and BMLF-1 specific T-cells or VZV IE62-A, IE62-B, and IE63 specific T-cells (p > 0.05). Representative ELISPOT cultures that illustrate the divergence of CMV-specific NK-cell and T-cell responses are shown in Fig. 2B. The effects of latent CMV infection and ‘CMV response’ category on NK-cell cytotoxic activity (NKCA) against the K562 cell line are described in Fig. 3A. There was a main effect of latent CMV infection on NKCA [F(1, 192) = 14.31, p < 0.001] that was independent
CMVpos NKlow Tlow [N = 4]
CMVneg [N = 24]
One-way ANOVA F statistic (p value)
(0.30) (0.14) (0.10) (0.92) (0.01)⁄
31 (<.001)⁄
(<.01)⁄ (<.01)⁄ (0.19) (0.74) (0.22)
13 (<.001)⁄
of NK-cell dose [F(3, 192) = 0.74, p = 0.529]. In addition, there was a main effect of ‘CMV response’ category on NKCA [F(3, 96) = 83.82, p < 0.001] that was driven by markedly increased NKCA in the NKhigh/Tlow group and decreased NKCA in the NKlow/Tlow group (p < 0.001). The effect of ‘CMV response’ category increased with increasing NK-cell dose [F(9, 96) = 4.48, p < 0.001]. Representative flow cytometry dot-plots that illustrate the effect of ‘CMV response’ category on NK-cell cytotoxicity are displayed in Fig. 3B.
4. Discussion Our study is the first to show a reciprocal relationship between CMV-driven accumulation of highly-differentiated NKG2C+ NKcells and CMV-specific T-cell responses. Subjects with effective CMV control (as suggested by low CMV IgG titers) had a high proportion of NKG2C+/CD57+ NK-cells or CD28 /CD27 CD8+ T-cells, but not both. Interestingly, an elevated proportion of NKG2C+/ CD57+ NK-cells coupled to a low proportion of CD27 /CD28 CD8+ T-cells (NKhigh/Tlow) was associated with a marked increase in NK-cell-mediated cytotoxic activity (NKCA) against the NKsensitive K562 cell line. Those with an NKhigh/Tlow phenotype also
Fig. 2. CMV-specific T-cell responses are uncoupled from CMV-driven accumulation of highly-differentiated NKG2C + NK-cells. (a) Shows the effect of CMV serostatus and ‘CMV response’ category on the number of CMV-pp65 and CMV-IE1 specific spot-forming cells (SFC) per 1 106 total T-cells in CMVpos subjects. Values are mean ± SE. A statistically greater proportion of CMV-specific T-cells based on CMV serostatus (CMVpos > CMVneg) or CMV response category (Thigh > Tlow) is indicated by #p < 0.001. (b) Displays representative ELISPOT cultures for CMV-infected subjects based on ‘CMV response’ category.
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had low CMV IgG titers despite having very few detectable IFN-c secreting CMV-specific T-cells. Further, CMV-infected subjects without a significant NK-cell or T-cell phenotype response (NKlow/ Tlow) had negligible CMV-specific T-cell responses, markedly decreased NKCA, and high CMV IgG titers. Overall, these findings indicate that individual responses to CMV infection are highly variable with effective viral control being achieved through increased differentiation and CMV-specific responses in either the CD8+ Tcell or NK-cell compartments, but not both. It has long been known that CMV, even in latency, is able to drive the preferential expansion of highly-differentiated NKG2C+ NK-cells [12] and CD8+ T-cells [9]. These CMV-driven responses, however, are highly variable between CMV-infected subjects [12] and it has been reported recently that the ‘‘CMV-specific” expansions of highly-differentiated NKG2C + NK-cells and CD8+ T-cells are uncoupled from one another [13]. We show here that there is in fact an inverse relationship between CMV-specific NK-cell and T-cell responses in those with low CMV IgG titers (<7 IU/mL). In other words, individuals with a high proportion of CD8+/CD27 / CD28 T-cells have a low percentage of NKG2C+/CD57 + NK-cells and vice versa. This reciprocal relationship between NK-cell and T-cell phenotypes appears to have profound functional implications. We have previously shown that latent CMV infection is associated with increased NKCA against the NK-sensitive K562 cell line [5], and marked clonal expansion of antigen-specific T-cells responding to a diverse array of CMV-associated peptides has been reported in CMV-positive individuals [9]. As with the divergence of NK-cell and T-cell phenotypic responses, strong NK-cell and T-cell functional responses are inversely related and not seen together in those with low CMV IgG titers. Overall, this data may have important implications for our understanding of the immune response to CMV and the diversity of mechanisms that the immune system can utilize to control CMV. It has long been thought that a large memory pool of CMVspecific T-cells was required to maintain CMV in a latent state [17]. It was hypothesized that the accumulation of CMV-specific T-cells over the lifespan was a major contributor to immunosenescence and the loss of TCR diversity with age [17]. We show here, however, that individuals with a ‘‘NK-cell only” response to CMV (NKhigh/Tlow) have markedly increased NKCA against the K562 cell line and CMV-specific T-cell responses that are indistinguishable from CMVneg subjects. On the other hand, individuals with a ‘‘Tcell only” response (NKlow/Thigh) have high CMV-specific T-cell responses to pp65 and IE1, a decreased proportion of naïve CD8+
T-cells (CD28+/CD27+), and NKCA against the K562 cell line that was comparable to CMVneg subjects. In the present cohort, 63% of individuals latently infected with CMV have either an NK-cell or T-cell response to CMV (NKhigh/Tlow or NKlow/Thigh), while only 20% have both (NKhigh/Thigh). Those subjects with a combined NK-cell/T-cell response (NKhigh/Thigh) have high CMV IgG titers, and NKCA and CMV-specific T-cell responses that are greater than CMVneg subjects, but less than ‘‘NK-cell only” (NKhigh/Tlow) or ‘‘Tcell only” (NKlow/Thigh) responders, respectively. Considering that a dichotomous response is associated with low Ab titers, it could be that the subjects with a dual response were unable to control the virus adequately with an initial NK-cell or T-cell mediated response, thus a second response was required. The NK-cell/Tcell functional dichotomy observed in those with effective CMV control may be due to the rheostat-like properties of NK-cells whereby activated NK-cells lyse viral-specific T-cells, thus preventing immune pathology secondary to hyperactive anti-viral T-cell responses [18]. This suggests that a vigorous NK-cell response may slow the progression of CMV-driven immunosenescence and autoimmunity by preventing the accumulation of CMV-specific and late-differentiated T-cells that can become functionally exhausted in old age [17]. Interestingly, those subjects without a significant NK-cell or T-cell phenotype response (NKlow/Tlow) had high CMV IgG titers, markedly decreased NKCA, and CMV-specific T-cell responses that were indistinguishable from those of CMVneg individuals. Thus, in a minority of subjects (17%), CMV is associated with inhibited NK-cell function and elicits no T-cell response at all. As the literature regarding the deleterious effects of CMV on Tcell phenotype and TCR diversity has evolved, researchers have postulated that it may be useful as a matter of public health to develop a vaccine against CMV [2,3]. It is thought that this would protect against acute CMV infection in the immunocompromised and also improve longevity in the elderly due to CMV’s inclusion in the so-called Immune Risk Profile [2]. Our findings suggest that this idea may be premature as ‘‘CMV-specific” expansion of NKG2C + NK-cells enhances NKCA and blocks excessive CMV-specific Tcell responses in those with CMV IgG titers below 7 IU/mL. In other studies, a high proportion of NKG2C+ NK-cells has been shown to prevent CMV reactivation [7,8] and expansion of NKG2C+ NKcells is strongly associated with resolution of active CMV infection [12]. Interestingly, these ‘‘CMV-specific” NK-cells also show evidence of immunological memory. While the typical lifespan of a non-proliferating NK cell is only 10 days [19], long-lived, ‘memory-like’ CMV-specific NK-cells have been identified in mice
Fig. 3. NK-cell cytotoxic activity (NKCA) is markedly increased in NKhigh/Tlow subjects and decreased in NKlow/Tlow CMVpos subjects. (a) Presents NKCA (%) against the K562 cell line at a 10:1 effector-to-target cell ratio based on CMV serostatus and ‘CMV response’ category. Values are mean ± SE. Statistically significant differences from NKlow/Tlow subjects are indicated by #p < 0.001 and statistically significant differences from all other ‘CMV response’ categories are indicated by ^p < 0.001. A statistically significant effect of CMV serostatus is denoted by *p < 0.001. (b) Displays representative flow cytometry dot-plots for the NK-cell cytotoxicity assay.
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and these cells are capable of generating recall responses and persisting for several months in lymphoid and non-lymphoid organs [20]. In humans, a stable phenotypic imprint of CMV on NK-cells (e.g. upregulation of NKG2C and certain inhibitory killer immunoglobulin-like receptors) can be maintained for years through homeostatic proliferation [6]. These observations have led to the view of NKG2C+ NK-cells as ‘‘memory” NK-cells [11]; however, unlike with memory T-cells, NKG2C+ NK-cells have broad specificity. For example, NKG2C+ NK-cells have been observed to expand in response to active Hantavirus, Chikungunya, HIV, and HBV infections, but only in individuals previously infected with CMV [21]. Thus, it may be more advantageous to strengthen CMV-specific NK-cell responses relative to T-cell responses rather than eradicating CMV or preventing its spread through vaccination. Such an approach, would likely enhance NK-cell function and broadly strengthen anti-viral immunity without jeopardizing immunological control of CMV. Interestingly, all CMVpos subjects in the NKhigh/Thigh and NKlow/Tlow groups had much higher CMV IgG antibody titers than the subjects in the NKhigh/Tlow and NKlow/ Thigh groups. This suggests that a combined response or no response at all might be evidence of poor CMV control (i.e. more frequent reactivation and boosting of the antibody response). Future studies should determine if these categories can predict risk of reactivated CMV infection in vulnerable populations or susceptibility to CMV-associated immunosenescence [2] and chronic diseases linked to high CMV IgG titers, such as coronary artery disease and atherosclerosis [22]. CMV-driven expansion of NKG2C+ NKcells may also have implications for cancer incidence as enhanced NKCA has been linked to a decreased risk of cancer [23]. Specifically, CMV-driven expansion of NKG2C+ NK-cells has the potential to improve immunity against any tumors or viruses that use upregulation of HLA-E as a means of immunoevasion. For example, clinical CMV reactivation and the subsequent accumulation of NKG2C+ NK-cells have been linked to a decreased risk of relapse in acute myeloid leukemia patients [24,25]. In addition, prior exposure to CMV is associated with increased anti-viral responses to viruses that upregulate HLA-E, such as Hantavirus [26]. Limitations of this study include the small sample size and fairly homogeneous subject population. Thus, future studies should determine the effects of these ‘CMV response’ categories on immune function, disease susceptibility, and longevity in a larger CMVpos cohort with a wider age distribution. Future studies should also investigate the effects of stress on CMV-specific NK-cell and Tcell responses as stress has been linked to immune dysfunction and high CMV-specific antibody titers [27]. We also acknowledge that measurement of CMV IgG antibody titers is a relatively course measure of viral control as recent evidence suggests that IgG titers do not correlate well with CMV DNA measurements in the elderly [28,29]. However, high CMV IgG concentration is still evidence of immune dysfunction as it has been linked to chronic inflammation [30], atherosclerosis [22], stress-induced immune decrements [27], and increased all-cause mortality in the elderly [31]. Future studies should determine if the present findings hold true when more sensitive markers of CMV control are used, such as the shedding of CMV DNA in bodily fluids and/or the presence of CMV DNA in blood monocytes [28,29]. It will also be important to confirm that the lack of a CMV-specific T-cell response observed in some subjects was not due to a skewing of the CMV-specific T-cell pool to other CMV antigens that were not measured here [32]. In conclusion, CMVpos individuals with effective viral control (low CMV IgG titers) can be separated into one of two groups: (1) NK-cell responders with an elevated NKG2C+ NK-cell proportion, low CMV-specific T-cell responses, and high NKCA or (2) T-cell responders with an elevated proportion of highlydifferentiated and CMV-specific CD8+ T-cells, low NKG2C+ NKcell proportion, and low NKCA. Conversely, those CMVpos subjects
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with a combined NK-cell/T-cell response or no response at all show signs of decreased immunity and poor viral control. The findings in this study challenge the dogma that CMV has generally negative effects on immune function, as CMV-driven accumulation of NKG2C+ NK-cells is associated with increased function in the NKcell compartment. Further, these findings could have major implications for our understanding of the aging immune system as the deleterious effects of CMV on the T-cell compartment, which have long been linked with immunosenescence [2], may be abrogated by the expansion of highly functional, rheostat-like NKG2C+ NKcells. Future studies investigating the effects of CMV on cancer, viral immunity, vaccine responses, and overall health must account for the diversity in the immune response to CMV as the virus’ effects on NK-cell and T-cell function are far from uniform. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this article. Acknowledgments This work was supported by NASA Grant NNX12AB48G to R.J. Simpson and NSBRI Grant PF04307 to A.B. Bigley. References [1] S.L. Bate, S.C. Dollard, M.J. Cannon, Cytomegalovirus seroprevalence in the United States: the national health and nutrition examination surveys, 1988– 2004, Clin. Infect. Dis. 50 (11) (2010) 1439–1447. [2] A. Wikby, B. Johansson, J. Olsson, S. Lofgren, B.O. Nilsson, F. Ferguson, Expansions of peripheral blood CD8 T-lymphocyte subpopulations and an association with cytomegalovirus seropositivity in the elderly: the Swedish NONA immune study, Exp. Gerontol. 37 (2–3) (2002) 445–453. [3] G. Pawelec, Immunosenescence: role of cytomegalovirus, Exp. Gerontol. 54 (2014) 1–5. [4] D.W. White, C.R. Keppel, S.E. Schneider, T.A. Reese, J. Coder, J.E. Payton, T.J. Ley, H.W. Virgin, T.A. Fehniger, Latent herpesvirus infection arms NK cells, Blood 115 (22) (2010) 4377–4383. [5] A.B. Bigley, K. Rezvani, M. Pistillo, J. Reed, N. Agha, H. Kunz, D.P. O’Connor, T. Sekine, C.M. Bollard, R.J. Simpson, Acute exercise preferentially redeploys NKcells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells. Part II: impact of latent cytomegalovirus infection and catecholamine sensitivity, Brain Behav. Immun. (2015). doi: 10.1016/j.bbi.2014.12.027 (Epub ahead of print). [6] V. Beziat, L.L. Liu, J.A. Malmbeg, M.A. Ivarsson, E. Sohlberg, A.T. Bjorklund, C. Retiere, E. Sverremark-Ekstrom, J. Traheme, P. Ljungman, P. Schaffer, D.A. Price, J. Trowsdale, J. Michaelsson, H.G. Ljunggren, K.J. Malmberg, NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs, Blood 121 (14) (2013) 2678–2688. [7] V.D. Kheav, M. Busson, C. Scieux, R. Peffault de Latour, G. Maki, P. Haas, M.C. Mazeron, M. Carmagnat, E. Masson, A. Xhaard, M. Robin, P. Ribaud, N. Dulphy, P. Loiseau, D. Charron, G. Socie, A. Toubert, H. Moins-Teisserenc, Favorable impact of natural killer cell reconstitution on chronic graft-versus-host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation, Haematologica 99 (2014) 1860–1867. [8] A. Rolle, J. Pollmann, E.M. Ewen, V.T.K. Le, A. Halenius, H. Hengel, A. Cerwenka, IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion, J. Clin. Invest. 124 (12) (2014) 5305–5316. [9] R.A. van Lier, I.J. ten Berge, L.E. Gamadia, Human CD8(+) T-cell differentiation in response to viruses, Nat. Rev. Immunol. 3 (12) (2003) 931–939. [10] N.K. Bjorkstrom, P. Riese, F. Heuts, S. Andersson, C. Fauriat, M.A. Ivarsson, A.T. Bjorklund, M. Flodstrom-Tullberg, J. Michaelsson, M.E. Rottenberg, C.A. Guzman, H.G. Ljunggren, K.J. Malmberg, Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education, Blood 116 (19) (2010) 3853–3864. [11] S. Lopez-Verges, J.M. Milush, B.S. Schwartz, M.J. Pando, J. Jarjoura, V.A. York, J.P. Houchins, S. Miller, S.M. Kang, P.J. Norris, D.F. Nixon, L.L. Lanier, Expansion of a unique CD57(+)NKG2Chi natural killer cell subset during acute human cytomegalovirus infection, Proc. Natl. Acad. Sci. U.S.A. 108 (36) (2011) 14725–14732. [12] M. Guma, A. Angulo, C. Vilches, N. Gomez-Lozano, N. Malats, M. Lopez-Botet, Imprint of human cytomegalovirus infection on the NK cell receptor repertoire, Blood 104 (12) (2004) 3664–3671. [13] M. Bengner, V. Beziat, J. Ernerudh, B. Nilsson, S. Lofgren, A. Wikby, K.J. Malmberg, J. Strindhall, Independent skewing of the T cell and NK cell compartments associated with cytomegalovirus infection suggests division of labor between innate and adaptive immunity, Age 36 (2014) 571–582.
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Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Research paper
Dichotomous effects of latent CMV infection on the phenotype and functional properties of CD8+ T-cells and NK-cells Austin B. Bigley a, Guillaume Spielmann a,b, Nadia Agha a, Daniel P. O’Connor a, Richard J. Simpson a,⇑ a b
Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, TX 77204, USA School of Kinesiology, Louisiana State University, 112 Long Fieldhouse, Baton Rouge, LA 70803, USA
a r t i c l e
i n f o
Article history: Received 9 October 2015 Revised 9 November 2015 Accepted 23 November 2015 Available online 24 November 2015 Keywords: NKG2C CD57 CD27 CD28 Cytotoxicity NKCA K562 pp65 IE1 Immunosenescence Aging Viral load
a b s t r a c t CMV markedly alters the phenotype and function of NK-cells and T-cells and has been linked to immunosenescence. We show here that subjects with effective CMV control (evidenced by low CMV IgG titers) have functional responses to CMV that are driven by either NKG2C+ NK-cells or CMVspecific T-cells (15 of 24 subjects), but not both. These data indicate that people with effective CMV control are either NK-cell or T-cell responders, and corroborates the idea that NK-cells have rheostat-like properties that regulate anti-viral T-cell responses. Whether or not lifelong CMV control through either NK-cell or T-cell responses have implications for immunosenescence remains to be determined. Ó 2016 Published by Elsevier Inc.
1. Introduction CMV is a prevalent b-herpesvirus that infects 50–80% of all adults in the United States [1]. Within the T-cell compartment, CMV is often considered to be an immunological burden that exerts mostly negative effects on immune status and overall health [2]. This has led to the perception that it may be beneficial to eradicate CMV or vaccinate against it as a matter of public health [3]. However, recent evidence suggests that latent herpesviruses (including CMV) may play a pivotal role in cancer immunosurveillance by ‘arming’ NK-cells to adequately destroy malignant target cells [4,5]. For example, NK-cells in mice with latent Murid herpesvirus 4 infection show increased Granzyme B expression, IFN-c production and cytotoxicity, which protects infected mice from a lethal lymphoma challenge [4]. Further, we have reported increased NK-cell-mediated cytotoxicity against HLA-E+ human tumor cell lines in healthy people previously exposed to CMV [5]. Thus, it may be that CMV exerts divergent effects on NK-cell and
⇑ Corresponding author. E-mail address: [email protected] (R.J. Simpson). http://dx.doi.org/10.1016/j.cellimm.2015.11.005 0008-8749/Ó 2016 Published by Elsevier Inc.
T-cell immunity, with beneficial effects being found primarily within the NK-cell compartment. The functional effects of latent CMV infection on NK-cells and Tcells have been linked to clonal expansion of ‘‘CMV-specific” NKcell and T-cell subsets [6]. Within the NK-cell compartment, expansion of NK-cells expressing the activating receptor NKG2C is associated with greater viral control [7] due to increased lysis of CMV-infected cells that express HLA-E, the ligand for NKG2C [8]. This clonal expansion is associated with increased differentiation, which can result in functional exhaustion and replicative senescence, particularly in the T-cell compartment [9]. Highlydifferentiated T-cells can be defined by loss of CD28 and CD27 expression and NK-cell differentiation can be defined by acquisition of the terminal differentiation marker CD57 [10]. It has long been known that CMV is able to drive the preferential expansion of highly-differentiated NKG2C+ NK-cells [11] and CD8+ T-cells [9]. These CMV-driven responses are highly variable between CMV-infected subjects [12] and the differentiation status of Tcells has been reported to be independent of NK-cell differentiation in those with CMV [13]. It remains to be seen, however, how CMVspecific T-cell responses correlate with those of NK-cells at a func-
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tional level. If CMV-specific NK-cell and T-cell functional responses are reciprocally related, it may be that CMV boosts immunity in some, while inhibiting it in others. It is essential, therefore, that we understand the different mechanisms by which CMV is controlled in the healthy host before we consider eradication of CMV, as such an approach may have unforeseen consequences for anti-viral and anti-tumor immunity. The primary aims of this study were twofold. First, we measured NK-cell and T-cell differentiation in CMVpos subjects and defined ‘CMV response’ categories based on the relative magnitude of NK-cell and T-cell differentiation. Second, we determined the effect of ‘CMV response’ category on NK-cell cytotoxicity, CMVspecific functional T-cell responses, and CMV IgG antibody titers. We showed for the first time that CMV-specific NK-cell and T-cell responses are inversely related in healthy subjects with effective CMV control. 2. Methods 2.1. Subjects Forty-eight healthy adults (50% CMVpos; age: 32.3 ± 4.6 yr) voluntarily participated in this study. Subjects were between the ages of 18 and 50 and not taking any immunomodulatory medication. Abstinence from alcohol, caffeine and vigorous physical activity 24 h prior to blood donation, as well as elimination of vitamin/ mineral supplementation at least 4 weeks prior to taking part in the study was required and confirmed verbally with the subjects on their arrival to the laboratory. All subjects provided written informed consent prior to participating in the study and the Committee for the Protection of Human Subjects at the University of Houston approved the protocol.
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controls, respectively. The PepMixes for each viral antigen were assessed in separate assays. Spot-forming cells were enumerated by Zellnet Consulting Inc. (Fort Lee, NJ, USA) and compared with input cell numbers to obtain the frequency of virus-reactive Tcells. The number of spot forming cells in each assay was adjusted for the total CD3+ T-cell count.
2.4. NK-cell cytotoxicity assay As we have described previously [14], monocyte-depleted lymphocytes were co-cultured with anti-CD71 (IgG1, Clone OKT-9) FITC-labeled K562 cells (1.0 105 cells) at 1:1, 2.5:1, 5:1, and 10:1 lymphocyte: K562 cell ratios in a final volume of 2.2 mL of 10% FBS-RPMI 1640. After 4 h incubation at 37 °C, the cells were washed and stained with PE-conjugated anti-CD3 (IgG1, Clone UCHT1) and APC-conjugated anti-CD56 (IgG1, Clone CMSSB) antibodies to quantify the number of NK-cells in each tube. After a final wash, propidium iodide (PI) was added and the number of NKcells, live target cells, and dead target cells was resolved using 4color flow cytometry [16]. All antibodies and the PI were purchased from eBioscience. The K562 cells were purchased from ATCC (Manassas, VA, USA).
2.5. CMV serology Fasting serum samples were frozen at 80 °C until measurement of CMV IgG antibodies, which were analyzed in duplicate using commercially available ELISA kits (BioCheck, Foster City, CA, USA) and a 96-well microplate reader (Molecular Devices, Sunnyvale, CA, USA). Antibody titers were calculated by comparing serum samples to references of known concentrations in accordance with the manufacturer’s instructions.
2.2. Flow cytometry Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Histopaque per the manufacturer’s instructions (Sigma–Aldrich, St. Louis, MO, USA). Aliquots of 1 106 isolated cells were incubated for 30 min with 50 lL of pre-diluted APC-conjugated anti-CD3 (IgG2a, Clone UCHT1), PECy5-conjugated anti-CD8 (IgG2a, Clone MEM-31) or PerCPeFluor710-conjugated anti-CD56 (IgG1, Clone CMSSB) or antiCD4 (IgG1, Clone SK3), Alexa488-conjugated anti-NKG2C (IgG1, Clone 134591) or anti-CD28 (IgG1, Clone CD28.2), and PEconjugated anti-CD57 (IgM, Clone TB01) or anti-CD27 (IgG1, Clone O323) monoclonal antibodies. The anti-CD56, anti-CD8, anti-CD4, anti-CD3, anti-CD57, anti-CD28, and anti-CD27 antibodies were purchased from eBioscience (San Diego, CA, USA) and the antiNKG2C antibody was purchased from R&D Systems (Minneapolis, MN, USA). Lymphocyte phenotype and cell count were assessed by 4-color flow cytometry using an Accuri C6 flow cytometer (Accuri, Ann Arbor, MI, USA) as previously described [14]. 2.3. Frequency and epitope specificity of CMV specific T-cells Enzyme-linked immunospot (ELISPOT) analysis was used to determine the frequency of T-cells secreting IFN-c in response to CMVpp65 or CMV IE-1 PepMixes (JPT Peptide Technologies, Berlin, Germany), which contain all 15mer peptides of each viral antigen in one pool [15]. T-cell responses to antigens of Eptein-Barr Virus (EBV) [Imp-2 and BMLF-1] and Varicella Zoster Virus (VZV) [IE62-A, IE62-B, and IE63] were also measured. ELISPOT assays were performed on PBMCs isolated in CMVpos donors. PBMCs that were unstimulated or stimulated with 1 lg/mL PHA (Sigma– Aldrich, St. Louis, MO, USA) served as negative and positive
2.6. Statistical analysis Data was statistically analyzed using the Predictive Analytics SoftWare (PASW 22.0) statistics computer program. CMVpos subjects (N = 24) were stratified based on their NK-cell and CD8+ Tcell differentiation responses. A high NK-cell differentiation response (NKhigh) was defined as an NKG2C+/CD57+ NK-cell proportion greater than the upper bound of the 95% confidence interval for CMVneg subjects, which was 13% of total NK-cells. A high Tcell differentiation response (Thigh) was defined as a CD28 /CD27 CD8+ T-cell proportion greater than the upper bound of the 95% confidence interval for CMVneg subjects, which was 13% of total CD8+ T-cells. Individuals with a proportion of differentiated NKcells and CD8+ T-cells less than the upper bound of the 95% confidence interval for CMVneg subjects (i.e. <13%) were defined as NKlow and Tlow, respectively. This allowed us to separate CMVpos subjects into 4 discrete response categories: (1) NKhigh/Tlow, (2) NKlow/Thigh, (3) NKhigh/Thigh, and (4) NKlow/Tlow. The effects of CMV serostatus and response category on the proportion and number of lymphocyte subsets were determined using independent sample t-tests and one-way ANOVA, respectively. To examine the effects of CMV serostatus and response category on NK-cell cytotoxicity, a maximum likelihood linear mixed model (LMM) was used that included main effects for dose (1X, 2.5X, 5X, or 10X) and CMV serostatus or ‘CMV response’ category as well as interaction effects of CMV serostatus x dose and ‘CMV response’ category x dose. Bonferroni post hoc analysis was performed to determine the locations of the significant effects for ‘CMV response’ category and dose. The upper bound of the 95% confidence interval was defined as the mean + (1.96 * standard deviation). Statistical significance was defined as p < 0.05.
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3. Results 3.1. Divergence of NK-cell and T-cell differentiation in response to latent CMV infection The effects of latent CMV infection and ‘CMV response’ category on the proportions of highly differentiated NKG2C + NK-cells and CD8+ T-cells are described in Fig. 1A. The proportions of NKG2C +/CD57 + NK-cells and CD28 /CD27 CD8+ T-cells were greater in CMVpos subjects (p < 0.001). Within the CMVpos cohort (N = 24), 6 were NKhigh/Tlow, 9 were NKlow/Thigh, 5 were NKhigh/Thigh, and 4 were NKlow/Tlow. There was no difference in age between these ‘CMV response’ categories (p > 0.05). Representative flow cytometry dot-plots that illustrate the divergence of NK-cell and T-cell differentiation during latent CMV infection are shown in Fig. 1B. The proportion of NKG2C+/CD57+ NK-cells was elevated in the NKhigh/ Tlow and NKhigh/Thigh groups (p < 0.001). The proportion of CD28 /
CD27 /CD8+ T-cells was increased and the proportion of ‘‘naïve” CD28+/CD27+/CD8+ T-cells was decreased in the NKlow/Thigh and NKhigh/Thigh groups (p < 0.001). Additionally, the proportion of CD28 /CD27 /CD4+ T-cells was increased in CMVpos subjects (p < 0.001), but there were no differences based on ‘CMV response’ category (p > 0.05). The individual NK-cell and T-cell differentiation responses of the 24 CMVpos subjects are shown in Fig. 1C. The effects of latent CMV infection and ‘CMV response’ category on individual NK-cell and T-cell subsets and CMV IgG antibody titers are presented in Table 1. The numbers of NKG2C+/CD57+ and NKG2C+/CD57 NK-cells were greater in the NKhigh/Tlow and NKhigh /Thigh groups (p < 0.001); the number of CD28 /CD27 CD8+ T-cells was greater in the NKlow/Thigh and NKhigh/Thigh groups (p < 0.001); the numbers of CD28+/CD27+ CD4+ and CD8+ T-cells, and total CD4+ T-cells were greater in the NKlow Tlow group (p < 0.05); and the number of total CD8+ T-cells was lower in the NKhigh/Tlow group (p < 0.01). CMV IgG antibody titers are elevated
Fig. 1. The CMV-driven accumulation of NKG2C+/CD57+ NK-cells is inversely related to that of CD8+/CD28 /CD27 T-cells in a majority of subjects. (a) Shows the proportions of NKG2C+/CD57 + NK-cells (% of total NK-cells) and CD8+/CD28 /CD27 T-cells (% of total CD8+ T-cells) based on CMV serostatus and ‘CMV response’ category. Values are mean ± SE. A statistically greater proportion of NKG2C+/CD57 + NK-cells or CD8+/CD28 /CD27 T-cells based on CMV serostatus (CMVpos > CMVneg) or CMV response category (e.g. NKhigh > NKlow, Thigh > Tlow) is indicated by #p < 0.001. (b) Displays representative flow cytometry dot-plots for co-expression of NKG2C with CD57 on NK-cells and CD28 with CD27 on CD8+ T-cells. (c) Presents the interaction between NK-cell and T-cell differentiation in individual CMVpos subjects (N = 24). The gray dashed line represents the upper bound of the 95% confidence interval for the CMVneg cohort (N = 24).
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Table 1 Lymphocyte subset numbers contrasted by CMV serostatus and CMV response category. Main effects among the nominal variables (CMV serostatus and CMV response category) are shown with significance indicated by *(p < 0.05). Statistical differences between CMV response categories are indicated by #(p < 0.05). Data are mean ± SD. CMVpos NKhigh Tlow [N = 6]
CMVpos NKlow Thigh [N = 9]
CMVpos NKhigh Thigh [N = 5]
CMV serostatus
CMV response category
NK-cells NKG2C+/CD57+ NKG2C+/CD57 NKG2C-/CD57+ NKG2C-/CD57
246.0 ± 77.9 96.7 ± 41.9# 30.8 ± 19.1# 58.4 ± 70.2 60.1 ± 24.3
201.0 ± 80.9 8.2 ± 6.5 6.2 ± 4.4 106.4 ± 58.3 80.2 ± 28.4
216.2 ± 54.6 66.0 ± 44.1# 22.5 ± 9.5# 72.7 ± 12.8 55.0 ± 20.7
194.4 ± 55.2 4.8 ± 1.6 8.2 ± 4.2 90.8 ± 63.1 90.6 ± 33.7
167.1 ± 80.0 5.7 ± 4.9 8.9 ± 7.3 67.8 ± 53.5 84.7 ± 46.4
3.8 (0.06) 13 (<.001)⁄ 4.8 (0.03)⁄ 2.6 (0.11) 1.8 (0.19)
0.6 (0.61) 17 (<.001)⁄ 7 (<.01)⁄ 0.3 (0.81) 2.5 (0.89)
CD8+ T-cells CD28+/CD27+ CD28+/CD27 CD28 /CD27+ CD28 /CD27
189.0 ± 15.8# 152.3 ± 16.5 10.0 ± 6.5 16.3 ± 8.1 10.3 ± 1.5
369.8 ± 105.7 228.3 ± 34.7 13.7 ± 12.2 64.7 ± 50.0 90.4 ± 34.6#
352.7 ± 98.3 186.0 ± 43.3 12.0 ± 9.6 50.5 ± 26.9 104.2 ± 36.7#
417.8 ± 88.4 328.5 ± 23.3# 6.8 ± 6.9 61.7 ± 71.6 20.8 ± 9.5
342.6 ± 161.4 285.4 ± 154.6 8.9 ± 7.3 34.2 ± 22.3 14.1 ± 11.6
0.2 (0.63) 1.5 (0.23) 0.8 (0.39) 1.5 (0.23) 17 (<.001)⁄
5.1 (<.01)⁄ 23 (<.001)⁄ 0.6 (0.63) 0.7 (0.54) 17 (<.001)⁄
CD4+ T-cells CD28+/CD27+ CD28+/CD27 CD28 /CD27+ CD28 /CD27
541.0 ± 115.6 508.3 ± 101.2 22.7 ± 2.8 2.3 ± 1.5 7.7 ± 12.4
681.3 ± 203.5 623.7 ± 212.6 41.5 ± 25.4 1.3 ± 1.0 14.8 ± 16.8
654.5 ± 181.3 574.0 ± 137.8 57.0 ± 28.7 2.8 ± 2.2 20.7 ± 7.0
1282.8 ± 376.5# 1172.8 ± 357.0# 74.8 ± 47.6 1.5 ± 1.2 33.7 ± 32.0
968.1 ± 381.2 923.1 ± 364.9 39.3 ± 19.8 4.8 ± 6.1 0.9 ± 1.6
1.1 2.3 2.8 0.1 7.5
7.2 7.9 1.7 0.4 1.6
2.6 ± 2.0
4.3 ± 2.1
17.2 ± 7.8#
16.0 ± 6.8#
0.3 ± 0.2
Cell count (cells/ll)
CMV IgG (IU/mL)
in subjects in the NKhigh/Thigh and NKlow/Tlow groups relative to those in the NKhigh/Tlow and NKlow/Thigh groups (p < 0.01). 3.2. CMV-specific T-cell responses are uncoupled from CMV-driven accumulation of highly-differentiated NKG2C+ NK-cells and NK-cellmediated cytotoxicity The effect of ‘CMV response’ category on the number of CMVspecific T-cells is described in Fig. 2A. The number of T-cells responding to the CMV pp65 antigen was greater in the NKlow/Thigh and NKhigh/Thigh groups (p < 0.01) and the number of T-cells responding to the CMV IE-1 antigen was greater in the NKlow/Thigh group (p < 0.05). All of the CMVpos subjects had viral-responsive cells for EBV and VZV, but there was no effect of ‘CMV response’ category on the number of EBV Imp-2 and BMLF-1 specific T-cells or VZV IE62-A, IE62-B, and IE63 specific T-cells (p > 0.05). Representative ELISPOT cultures that illustrate the divergence of CMV-specific NK-cell and T-cell responses are shown in Fig. 2B. The effects of latent CMV infection and ‘CMV response’ category on NK-cell cytotoxic activity (NKCA) against the K562 cell line are described in Fig. 3A. There was a main effect of latent CMV infection on NKCA [F(1, 192) = 14.31, p < 0.001] that was independent
CMVpos NKlow Tlow [N = 4]
CMVneg [N = 24]
One-way ANOVA F statistic (p value)
(0.30) (0.14) (0.10) (0.92) (0.01)⁄
31 (<.001)⁄
(<.01)⁄ (<.01)⁄ (0.19) (0.74) (0.22)
13 (<.001)⁄
of NK-cell dose [F(3, 192) = 0.74, p = 0.529]. In addition, there was a main effect of ‘CMV response’ category on NKCA [F(3, 96) = 83.82, p < 0.001] that was driven by markedly increased NKCA in the NKhigh/Tlow group and decreased NKCA in the NKlow/Tlow group (p < 0.001). The effect of ‘CMV response’ category increased with increasing NK-cell dose [F(9, 96) = 4.48, p < 0.001]. Representative flow cytometry dot-plots that illustrate the effect of ‘CMV response’ category on NK-cell cytotoxicity are displayed in Fig. 3B.
4. Discussion Our study is the first to show a reciprocal relationship between CMV-driven accumulation of highly-differentiated NKG2C+ NKcells and CMV-specific T-cell responses. Subjects with effective CMV control (as suggested by low CMV IgG titers) had a high proportion of NKG2C+/CD57+ NK-cells or CD28 /CD27 CD8+ T-cells, but not both. Interestingly, an elevated proportion of NKG2C+/ CD57+ NK-cells coupled to a low proportion of CD27 /CD28 CD8+ T-cells (NKhigh/Tlow) was associated with a marked increase in NK-cell-mediated cytotoxic activity (NKCA) against the NKsensitive K562 cell line. Those with an NKhigh/Tlow phenotype also
Fig. 2. CMV-specific T-cell responses are uncoupled from CMV-driven accumulation of highly-differentiated NKG2C + NK-cells. (a) Shows the effect of CMV serostatus and ‘CMV response’ category on the number of CMV-pp65 and CMV-IE1 specific spot-forming cells (SFC) per 1 106 total T-cells in CMVpos subjects. Values are mean ± SE. A statistically greater proportion of CMV-specific T-cells based on CMV serostatus (CMVpos > CMVneg) or CMV response category (Thigh > Tlow) is indicated by #p < 0.001. (b) Displays representative ELISPOT cultures for CMV-infected subjects based on ‘CMV response’ category.
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had low CMV IgG titers despite having very few detectable IFN-c secreting CMV-specific T-cells. Further, CMV-infected subjects without a significant NK-cell or T-cell phenotype response (NKlow/ Tlow) had negligible CMV-specific T-cell responses, markedly decreased NKCA, and high CMV IgG titers. Overall, these findings indicate that individual responses to CMV infection are highly variable with effective viral control being achieved through increased differentiation and CMV-specific responses in either the CD8+ Tcell or NK-cell compartments, but not both. It has long been known that CMV, even in latency, is able to drive the preferential expansion of highly-differentiated NKG2C+ NK-cells [12] and CD8+ T-cells [9]. These CMV-driven responses, however, are highly variable between CMV-infected subjects [12] and it has been reported recently that the ‘‘CMV-specific” expansions of highly-differentiated NKG2C + NK-cells and CD8+ T-cells are uncoupled from one another [13]. We show here that there is in fact an inverse relationship between CMV-specific NK-cell and T-cell responses in those with low CMV IgG titers (<7 IU/mL). In other words, individuals with a high proportion of CD8+/CD27 / CD28 T-cells have a low percentage of NKG2C+/CD57 + NK-cells and vice versa. This reciprocal relationship between NK-cell and T-cell phenotypes appears to have profound functional implications. We have previously shown that latent CMV infection is associated with increased NKCA against the NK-sensitive K562 cell line [5], and marked clonal expansion of antigen-specific T-cells responding to a diverse array of CMV-associated peptides has been reported in CMV-positive individuals [9]. As with the divergence of NK-cell and T-cell phenotypic responses, strong NK-cell and T-cell functional responses are inversely related and not seen together in those with low CMV IgG titers. Overall, this data may have important implications for our understanding of the immune response to CMV and the diversity of mechanisms that the immune system can utilize to control CMV. It has long been thought that a large memory pool of CMVspecific T-cells was required to maintain CMV in a latent state [17]. It was hypothesized that the accumulation of CMV-specific T-cells over the lifespan was a major contributor to immunosenescence and the loss of TCR diversity with age [17]. We show here, however, that individuals with a ‘‘NK-cell only” response to CMV (NKhigh/Tlow) have markedly increased NKCA against the K562 cell line and CMV-specific T-cell responses that are indistinguishable from CMVneg subjects. On the other hand, individuals with a ‘‘Tcell only” response (NKlow/Thigh) have high CMV-specific T-cell responses to pp65 and IE1, a decreased proportion of naïve CD8+
T-cells (CD28+/CD27+), and NKCA against the K562 cell line that was comparable to CMVneg subjects. In the present cohort, 63% of individuals latently infected with CMV have either an NK-cell or T-cell response to CMV (NKhigh/Tlow or NKlow/Thigh), while only 20% have both (NKhigh/Thigh). Those subjects with a combined NK-cell/T-cell response (NKhigh/Thigh) have high CMV IgG titers, and NKCA and CMV-specific T-cell responses that are greater than CMVneg subjects, but less than ‘‘NK-cell only” (NKhigh/Tlow) or ‘‘Tcell only” (NKlow/Thigh) responders, respectively. Considering that a dichotomous response is associated with low Ab titers, it could be that the subjects with a dual response were unable to control the virus adequately with an initial NK-cell or T-cell mediated response, thus a second response was required. The NK-cell/Tcell functional dichotomy observed in those with effective CMV control may be due to the rheostat-like properties of NK-cells whereby activated NK-cells lyse viral-specific T-cells, thus preventing immune pathology secondary to hyperactive anti-viral T-cell responses [18]. This suggests that a vigorous NK-cell response may slow the progression of CMV-driven immunosenescence and autoimmunity by preventing the accumulation of CMV-specific and late-differentiated T-cells that can become functionally exhausted in old age [17]. Interestingly, those subjects without a significant NK-cell or T-cell phenotype response (NKlow/Tlow) had high CMV IgG titers, markedly decreased NKCA, and CMV-specific T-cell responses that were indistinguishable from those of CMVneg individuals. Thus, in a minority of subjects (17%), CMV is associated with inhibited NK-cell function and elicits no T-cell response at all. As the literature regarding the deleterious effects of CMV on Tcell phenotype and TCR diversity has evolved, researchers have postulated that it may be useful as a matter of public health to develop a vaccine against CMV [2,3]. It is thought that this would protect against acute CMV infection in the immunocompromised and also improve longevity in the elderly due to CMV’s inclusion in the so-called Immune Risk Profile [2]. Our findings suggest that this idea may be premature as ‘‘CMV-specific” expansion of NKG2C + NK-cells enhances NKCA and blocks excessive CMV-specific Tcell responses in those with CMV IgG titers below 7 IU/mL. In other studies, a high proportion of NKG2C+ NK-cells has been shown to prevent CMV reactivation [7,8] and expansion of NKG2C+ NKcells is strongly associated with resolution of active CMV infection [12]. Interestingly, these ‘‘CMV-specific” NK-cells also show evidence of immunological memory. While the typical lifespan of a non-proliferating NK cell is only 10 days [19], long-lived, ‘memory-like’ CMV-specific NK-cells have been identified in mice
Fig. 3. NK-cell cytotoxic activity (NKCA) is markedly increased in NKhigh/Tlow subjects and decreased in NKlow/Tlow CMVpos subjects. (a) Presents NKCA (%) against the K562 cell line at a 10:1 effector-to-target cell ratio based on CMV serostatus and ‘CMV response’ category. Values are mean ± SE. Statistically significant differences from NKlow/Tlow subjects are indicated by #p < 0.001 and statistically significant differences from all other ‘CMV response’ categories are indicated by ^p < 0.001. A statistically significant effect of CMV serostatus is denoted by *p < 0.001. (b) Displays representative flow cytometry dot-plots for the NK-cell cytotoxicity assay.
A.B. Bigley et al. / Cellular Immunology 300 (2016) 26–32
and these cells are capable of generating recall responses and persisting for several months in lymphoid and non-lymphoid organs [20]. In humans, a stable phenotypic imprint of CMV on NK-cells (e.g. upregulation of NKG2C and certain inhibitory killer immunoglobulin-like receptors) can be maintained for years through homeostatic proliferation [6]. These observations have led to the view of NKG2C+ NK-cells as ‘‘memory” NK-cells [11]; however, unlike with memory T-cells, NKG2C+ NK-cells have broad specificity. For example, NKG2C+ NK-cells have been observed to expand in response to active Hantavirus, Chikungunya, HIV, and HBV infections, but only in individuals previously infected with CMV [21]. Thus, it may be more advantageous to strengthen CMV-specific NK-cell responses relative to T-cell responses rather than eradicating CMV or preventing its spread through vaccination. Such an approach, would likely enhance NK-cell function and broadly strengthen anti-viral immunity without jeopardizing immunological control of CMV. Interestingly, all CMVpos subjects in the NKhigh/Thigh and NKlow/Tlow groups had much higher CMV IgG antibody titers than the subjects in the NKhigh/Tlow and NKlow/ Thigh groups. This suggests that a combined response or no response at all might be evidence of poor CMV control (i.e. more frequent reactivation and boosting of the antibody response). Future studies should determine if these categories can predict risk of reactivated CMV infection in vulnerable populations or susceptibility to CMV-associated immunosenescence [2] and chronic diseases linked to high CMV IgG titers, such as coronary artery disease and atherosclerosis [22]. CMV-driven expansion of NKG2C+ NKcells may also have implications for cancer incidence as enhanced NKCA has been linked to a decreased risk of cancer [23]. Specifically, CMV-driven expansion of NKG2C+ NK-cells has the potential to improve immunity against any tumors or viruses that use upregulation of HLA-E as a means of immunoevasion. For example, clinical CMV reactivation and the subsequent accumulation of NKG2C+ NK-cells have been linked to a decreased risk of relapse in acute myeloid leukemia patients [24,25]. In addition, prior exposure to CMV is associated with increased anti-viral responses to viruses that upregulate HLA-E, such as Hantavirus [26]. Limitations of this study include the small sample size and fairly homogeneous subject population. Thus, future studies should determine the effects of these ‘CMV response’ categories on immune function, disease susceptibility, and longevity in a larger CMVpos cohort with a wider age distribution. Future studies should also investigate the effects of stress on CMV-specific NK-cell and Tcell responses as stress has been linked to immune dysfunction and high CMV-specific antibody titers [27]. We also acknowledge that measurement of CMV IgG antibody titers is a relatively course measure of viral control as recent evidence suggests that IgG titers do not correlate well with CMV DNA measurements in the elderly [28,29]. However, high CMV IgG concentration is still evidence of immune dysfunction as it has been linked to chronic inflammation [30], atherosclerosis [22], stress-induced immune decrements [27], and increased all-cause mortality in the elderly [31]. Future studies should determine if the present findings hold true when more sensitive markers of CMV control are used, such as the shedding of CMV DNA in bodily fluids and/or the presence of CMV DNA in blood monocytes [28,29]. It will also be important to confirm that the lack of a CMV-specific T-cell response observed in some subjects was not due to a skewing of the CMV-specific T-cell pool to other CMV antigens that were not measured here [32]. In conclusion, CMVpos individuals with effective viral control (low CMV IgG titers) can be separated into one of two groups: (1) NK-cell responders with an elevated NKG2C+ NK-cell proportion, low CMV-specific T-cell responses, and high NKCA or (2) T-cell responders with an elevated proportion of highlydifferentiated and CMV-specific CD8+ T-cells, low NKG2C+ NKcell proportion, and low NKCA. Conversely, those CMVpos subjects
31
with a combined NK-cell/T-cell response or no response at all show signs of decreased immunity and poor viral control. The findings in this study challenge the dogma that CMV has generally negative effects on immune function, as CMV-driven accumulation of NKG2C+ NK-cells is associated with increased function in the NKcell compartment. Further, these findings could have major implications for our understanding of the aging immune system as the deleterious effects of CMV on the T-cell compartment, which have long been linked with immunosenescence [2], may be abrogated by the expansion of highly functional, rheostat-like NKG2C+ NKcells. Future studies investigating the effects of CMV on cancer, viral immunity, vaccine responses, and overall health must account for the diversity in the immune response to CMV as the virus’ effects on NK-cell and T-cell function are far from uniform. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this article. Acknowledgments This work was supported by NASA Grant NNX12AB48G to R.J. Simpson and NSBRI Grant PF04307 to A.B. Bigley. References [1] S.L. Bate, S.C. Dollard, M.J. Cannon, Cytomegalovirus seroprevalence in the United States: the national health and nutrition examination surveys, 1988– 2004, Clin. Infect. Dis. 50 (11) (2010) 1439–1447. [2] A. Wikby, B. Johansson, J. Olsson, S. Lofgren, B.O. Nilsson, F. Ferguson, Expansions of peripheral blood CD8 T-lymphocyte subpopulations and an association with cytomegalovirus seropositivity in the elderly: the Swedish NONA immune study, Exp. Gerontol. 37 (2–3) (2002) 445–453. [3] G. Pawelec, Immunosenescence: role of cytomegalovirus, Exp. Gerontol. 54 (2014) 1–5. [4] D.W. White, C.R. Keppel, S.E. Schneider, T.A. Reese, J. Coder, J.E. Payton, T.J. Ley, H.W. Virgin, T.A. Fehniger, Latent herpesvirus infection arms NK cells, Blood 115 (22) (2010) 4377–4383. [5] A.B. Bigley, K. Rezvani, M. Pistillo, J. Reed, N. Agha, H. Kunz, D.P. O’Connor, T. Sekine, C.M. Bollard, R.J. Simpson, Acute exercise preferentially redeploys NKcells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells. Part II: impact of latent cytomegalovirus infection and catecholamine sensitivity, Brain Behav. Immun. (2015). doi: 10.1016/j.bbi.2014.12.027 (Epub ahead of print). [6] V. Beziat, L.L. Liu, J.A. Malmbeg, M.A. Ivarsson, E. Sohlberg, A.T. Bjorklund, C. Retiere, E. Sverremark-Ekstrom, J. Traheme, P. Ljungman, P. Schaffer, D.A. Price, J. Trowsdale, J. Michaelsson, H.G. Ljunggren, K.J. Malmberg, NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs, Blood 121 (14) (2013) 2678–2688. [7] V.D. Kheav, M. Busson, C. Scieux, R. Peffault de Latour, G. Maki, P. Haas, M.C. Mazeron, M. Carmagnat, E. Masson, A. Xhaard, M. Robin, P. Ribaud, N. Dulphy, P. Loiseau, D. Charron, G. Socie, A. Toubert, H. Moins-Teisserenc, Favorable impact of natural killer cell reconstitution on chronic graft-versus-host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation, Haematologica 99 (2014) 1860–1867. [8] A. Rolle, J. Pollmann, E.M. Ewen, V.T.K. Le, A. Halenius, H. Hengel, A. Cerwenka, IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion, J. Clin. Invest. 124 (12) (2014) 5305–5316. [9] R.A. van Lier, I.J. ten Berge, L.E. Gamadia, Human CD8(+) T-cell differentiation in response to viruses, Nat. Rev. Immunol. 3 (12) (2003) 931–939. [10] N.K. Bjorkstrom, P. Riese, F. Heuts, S. Andersson, C. Fauriat, M.A. Ivarsson, A.T. Bjorklund, M. Flodstrom-Tullberg, J. Michaelsson, M.E. Rottenberg, C.A. Guzman, H.G. Ljunggren, K.J. Malmberg, Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education, Blood 116 (19) (2010) 3853–3864. [11] S. Lopez-Verges, J.M. Milush, B.S. Schwartz, M.J. Pando, J. Jarjoura, V.A. York, J.P. Houchins, S. Miller, S.M. Kang, P.J. Norris, D.F. Nixon, L.L. Lanier, Expansion of a unique CD57(+)NKG2Chi natural killer cell subset during acute human cytomegalovirus infection, Proc. Natl. Acad. Sci. U.S.A. 108 (36) (2011) 14725–14732. [12] M. Guma, A. Angulo, C. Vilches, N. Gomez-Lozano, N. Malats, M. Lopez-Botet, Imprint of human cytomegalovirus infection on the NK cell receptor repertoire, Blood 104 (12) (2004) 3664–3671. [13] M. Bengner, V. Beziat, J. Ernerudh, B. Nilsson, S. Lofgren, A. Wikby, K.J. Malmberg, J. Strindhall, Independent skewing of the T cell and NK cell compartments associated with cytomegalovirus infection suggests division of labor between innate and adaptive immunity, Age 36 (2014) 571–582.
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