Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jun 26:10:275.
doi: 10.3389/fcimb.2020.00275. eCollection 2020.

Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay

Charlotte J Houldcroft  1 , Sarah E Jackson  1 , Eleanor Y Lim  1 , George X Sedikides  1 , Emma L Davies  1 , Claire Atkinson  2 , Megan McIntosh  2 , Ester B M Remmerswaal  3   4 , Georgina Okecha  1 , Frederike J Bemelman  3   4 , Richard J Stanton  5 , Matthew Reeves  2 , Mark R Wills  1
Affiliations

Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay

Charlotte J Houldcroft et al. Front Cell Infect Microbiol. .

Abstract

HCMV infection, reinfection or reactivation occurs in 60% of untreated solid organ transplant (SOT) recipients. Current clinical approaches to HCMV management include pre-emptive and prophylactic antiviral treatment strategies. The introduction of immune monitoring to better stratify patients at risk of viraemia and HCMV mediated disease could improve clinical management. Current approaches quantify T cell IFNγ responses specific for predominantly IE and pp65 proteins ex vivo, as a proxy for functional control of HCMV in vivo. However, these approaches have only a limited predictive ability. We measured the IFNγ T cell responses to an expanded panel of overlapping peptide pools specific for immunodominant HCMV proteins IE1/2, pp65, pp71, gB, UL144, and US3 in a cohort of D+R- kidney transplant recipients in a longitudinal analysis. Even with this increased antigen diversity, the results show that while all patients had detectable T cell responses, this did not correlate with control of HCMV replication in some. We wished to develop an assay that could directly measure anti-HCMV cell-mediated immunity. We evaluated three approaches, stimulation of PBMC with (i) whole HCMV lysate or (ii) a defined panel of immunodominant HCMV peptides, or (iii) fully autologous infected cells co-cultured with PBMC or isolated CD8+ T cells or NK cells. Stimulation with HCMV lysate often generated non-specific antiviral responses while stimulation with immunodominant HCMV peptide pools produced responses which were not necessarily antiviral despite strong IFNγ production. We demonstrated that IFNγ was only a minor component of secreted antiviral activity. Finally, we used an antiviral assay system to measure the effect of whole PBMC, and isolated CD8+ T cells and NK cells to control HCMV in infected autologous dermal fibroblasts. The results show that both PBMC and especially CD8+ T cells from HCMV seropositive donors have highly specific antiviral activity against HCMV. In addition, we were able to show that NK cells were also antiviral, but the level of this control was highly variable between donors and not dependant on HCMV seropositivity. Using this approach, we show that non-viraemic D+R+ SOT recipients had significant and specific antiviral activity against HCMV.

Keywords: T cells; antiviral; cell-mediated immunity; cytomegalovirus; herpesvirus; host-pathogen interactions; secreted immunity; transplantation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Analysis of longitudinal HCMV virus load and HCMV specific CD3+ T cell IFNγ responses of D+R– Kidney transplant patients. Two example D+R– kidney transplant patients with primary HCMV infection, T cell responses (spot forming units per 106 CD3+ T cells) were measured by IFNγ FluoroSpot (green triangles connected by a solid line) to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal T cell stimulation as a positive control (“POS”). Virus load (copies/ml blood) was measured by QNAT of HCMV DNA (pink hexagons connected by a dashed line). Cyan lines show the mean magnitude of response (±standard error) of CD3+ T cell IFNγ responses seen in healthy seropositive individuals in the same age decade as the transplant recipient for each peptide pool (Jackson et al., 2017b). (A) Patient 365 is an example of a D+R– patient with resolution of DNAemia following the emergence of detectable CD3+ IFNγ responses to four HCMV lytic peptide pools. (B) Patient 352, in contrast, had DNAemia which recurred several times, despite also developing detectable HCMV specific CD3+ T cell IFNγ responses which are comparable in frequency to those seen age-matched in healthy seropositives.
Figure 2
Figure 2
Quantification of HCMV dissemination and antiviral effect of polyclonally stimulated T cells. (A) Viral dissemination assay using a dual-fluorescently tagged HCMV strain (Merlin mCherry-P2A-UL36 [vICA], GFP-UL32 [pp150]). Following a low MOI (0.01) infection of indicator fibroblasts, infected cells become mCherry+ as the virus enters the immediate-early life cycle, and later become mCherry+ GFP+ as the virus enters the late life cycle. The percentage of cells infected can be visualized by fluorescent microscopy and quantified by two color flow cytometry. (B) Kinetics of virus dissemination in HFFF cells. Virus dissemination has been quantified by flow cytometry at various time points post-infection based on mCherry+ GFP– cells and on mCherry+ GFP+ cells. (C) Analysis of antiviral activity of supernatant from anti-CD3/CD28 stimulated T cells in PBMC (supernatant donor ARIA060). Fibroblasts were infected at 0.01 MOI and co-cultured with dilutions of supernatant, following 10 days of incubation fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry. (D) Analysis of antiviral activity of supernatant from anti-CD3/CD28 stimulated T cells in PBMC from 10 independent donors, diluted 1:4. Fibroblasts were infected at 0.01 MOI and co-cultured with dilutions of supernatant, following 9–11 days of incubation fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry.
Figure 3
Figure 3
Analysis of the antiviral activity of PBMC stimulated with HCMV-infected fibroblast lysates and pools of HCMV synthetic peptides specific for pp65, UL144,Gb, IE1,IE2, pp71, and US3. (A) Antiviral activity of supernatants derived from PBMC from three different donors stimulated with HCMV infected or uninfected fibroblast lysates. PBMC were also stimulated with anti-CD3/CD28 antibodies to generate a positive control antiviral supernatant. Fibroblasts were infected at 0.01 MOI and co-cultured with 1:4 dilution of supernatant, following 9–11 days of incubation fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry. Significance determined was by one-tailed T-test, p < 0.05. (B) Antiviral activity of HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control on five independent HCMV seropositive donors. Significance determined by one-tailed T-test. Key: *p < 0.05;**p < 0.005;***p < 0.0005.
Figure 4
Figure 4
Analysis of HCMV specific IFNγ FluoroSpot responses and antiviral activity of HCMV peptide stimulated supernatants with and without IFNγ depletion. (A) IFNγ FluoroSpot responses to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control of PBMC from donor CMV1801, calculated as spot-forming units (SFU) per 106 PBMC (background corrected). (B) IFNγ FluoroSpot responses to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control of PBMC from donor CMV332, calculated as spot-forming units (SFU) per 106 PBMC (background corrected). (C) The IFNγ concentration of supernatants following peptide stimulation (black) or after IFNγ depletion by anti-IFNγ-coated FluoroSpot (cyan), measured by ELISA. (D) The effect of IFNγ depletion on the antiviral activity of PBMC from donor CMV1801 stimulated with HCMV peptide pools for pp65/UL144, IE1&2, and pp71/US3 or anti-CD3/CD28 antibody. Bars labeled “IFNγ deplete” were harvested from anti-IFNγ antibody-coated FluoroSpot plates and added to a VDA in parallel with supernatants generated with the same stimulants and PBMC cell number. Significance determined by one-tailed T-test. Key: *p < 0.05;**p < 0.005;***p < 0.0005; ns, Not significant.
Figure 5
Figure 5
Antiviral activity of whole PBMC from HCMV seropositive and seronegative donors co-cultured with HCMV infected autologous fibroblasts. Violin plots showing results from viral dissemination assays of PBMC co-cultured with HCMV infected fibroblasts for 10–14 days over a range of E:T ratios, fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry. (A) Virus spread determined by mCherry+ fibroblasts and (B) by mCherry+ GFP+ fibroblasts. Cyan points show the range of control at each E:T for seropositive donors; magenta points are seronegative donors. Significance was determined by one-tailed T-test. Key: *p < 0.05;**p < 0.005;***p < 0.0005.
Figure 6
Figure 6
Antiviral activity of purified CD8+ T cells from HCMV seropositive and seronegative donors co-cultured with HCMV infected autologous fibroblasts. Violin plots showing results from Viral dissemination assays of PBMC co-cultured with HCMV infected fibroblasts for 10–14 days over a range of E:T ratios, fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry. (A) Virus spread determined by mCherry+ fibroblasts and (B) by mCherry+ GFP+ fibroblasts. Cyan points show the range of control at each E:T for seropositive donors; magenta points are seronegative donors. Significance was determined by one-tailed T-test. Key: *p < 0.05;**p < 0.005;***p < 0.0005.
Figure 7
Figure 7
Antiviral activity of purified NK cells from HCMV seropositive and seronegative donors co-cultured with HCMV infected autologous fibroblasts. Violin plots showing results from Viral dissemination assays of PBMC co-cultured with HCMV infected fibroblasts for 10–14 days over a range of E:T ratios, fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry. (A) Virus spread determined by mCherry+ fibroblasts and (B) by mCherry+ GFP+ fibroblasts. Cyan points show the range of control at each E:T for seropositive donors; magenta points are seronegative donors. Significance was determined by one-tailed T-test. Key: *p < 0.05;**p < 0.005;***p < 0.0005.
Figure 8
Figure 8
Antiviral activity of PBMC, CD8+ T cells, and NK cells from HCMV seropositive and seronegative donors and from non-viraemic D+R+ kidney transplant recipients co-cultured with HCMV infected autologous fibroblasts. Violin plots showing results from Viral dissemination assays of (A) PBMC, (B) CD8+ T cells, and (C) NK cells. Cells were co-cultured with HCMV infected fibroblasts for 10–14 days over a range of E:T ratios, fibroblasts were harvested and analyzed for mCherry and GFP expression by flow cytometry. Virus spread determined by mCherry+ fibroblasts and by mCherry+ GFP+ fibroblasts. D+R+ kidney transplant recipients were tested in a VDA from samples collected immediately pre-transplant (T1), at 1–2 months (T2), and 2 months post-transplant. At T3 (post-transplant), PBMC, CD8+ T cells, and NK cells were not statistically significantly different from healthy seropositives in their control of virus dissemination. Cyan points show the range of control at each E:T for seropositive donors; magenta points are seronegative donors. Purple points are samples taken immediately pre-transplant. Blue points are samples taken 1–2 months post-transplant. Gray points are samples taken 3 months post-transplant. Significance was determined by one-tailed T-test. Key: *p < 0.05;**p < 0.005;***p < 0.0005.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abate D., Saldan A., Fiscon M., Cofano S., Paciolla A., Furian L., et al. (2010). Evaluation of cytomegalovirus (CMV)-specific T cell immune reconstitution revealed that baseline antiviral immunity, prophylaxis, or preemptive therapy but not antithymocyte globulin treatment contribute to CMV-specific T cell reconstitution in kidney transplant recipients. J. Infect. Dis. 202, 585–594. 10.1086/654931 - DOI - PubMed
    1. Atabani S. F., Smith C., Atkinson C., Aldridge R. W., Rodriguez-Perálvarez M., Rolando N., et al. (2012). Cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy. Am. J. Transplant. 12, 2457–2464. 10.1111/j.1600-6143.2012.04087.x - DOI - PMC - PubMed
    1. Banas B., Böger C. A., Lückhoff G., Krüger B., Barabas S., Batzilla J., et al. . (2017). Validation of T-track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients. BMC Immunol. 18:15. 10.1186/s12865-017-0194-z - DOI - PMC - PubMed
    1. Baraniak I., Kropff B., Ambrose L., McIntosh M., McLean G. R., Pichon S., et al. . (2018). Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies. Proc. Natl. Acad. Sci. U.S.A. 115, 6273–6278. 10.1073/pnas.1800224115 - DOI - PMC - PubMed
    1. Becker J., Kinast V., Döring M., Lipps C., Duran V., Spanier J., et al. . (2018). Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines. Virulence 9, 1669–1684. 10.1080/21505594.2018.1535785 - DOI - PMC - PubMed

Publication types

Substances