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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Virology. 2020 May 10;547:1–6. doi: 10.1016/j.virol.2020.05.001

UNDERSTANDING THE MECHANISM OF ACTION OF CYTOMEGALOVIRUS-INDUCED REGULATORY T CELLS

Adriana Tovar-Salazar 1, Adriana Weinberg 1,*
PMCID: PMC7315853  NIHMSID: NIHMS1595803  PMID: 32442104

Abstract

We previously showed that CMV-induced CD4+CD27-CD28- T cells have regulatory (Treg) function. Here we sought to identify the target/s and the mechanistic underpinning/s of this effect. CMV-induced CD4+CD27-CD28- were sorted from CMV-stimulated PBMC and added to CMV-stimulated autologous PBMC cultures. Transwell experiments showed that the CMV-induced Treg mechanism of action required cell-to-cell contact. CMV-Treg significantly decreased proliferation of autologous CMV-stimulated CD8+ and, to a lesser extent, CD4+ T cells; reduced activation and increased apoptosis of CD4+ and CD8+ T cells; and increased apoptosis and expression of CTLA-4, T cell-inhibitory ligand, on dendritic cells. There was no effect on monocytes. Anti-PD-1, but not anti-CTLA-4, mAb-treatment increased proliferation of CD8+ T cells and decreased apoptosis of CD4+ and CD8+ T cells. Our data indicated that CD8+ T cells were the main target of CMV-specific Treg, which induced apoptosis of their targets using the PD-1 pathway.

Keywords: human cytomegalovirus, regulatory T cells, cell-mediated immunity

Introduction

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects 40 to 99% of adults depending on the geographic location and socio-economic status1,2. Although CMV infection is asymptomatic in most individuals, the virus has a profound effect on the immune system of infected individuals, including CMV-specific T cell inflation and expansion of NKG2C+ cells and of CD28- T cells3–8. Symptomatic CMV infection most frequently affects individuals with underlying cellular immune defects, such as older adults, transplant recipients and people with HIV9,10. In these individuals, CMV infection can amplify the risk of adverse outcomes through its own immune suppressive effects, which include decreased responses to vaccines and increased mortality in older adults; increased mortality unrelated to CMV end-organ disease in people with AIDS; faster progression to AIDS in CMV and HIV co-infected neonates; and increased risk of bacterial and fungal superinfections in transplant recipients9,11–15. High proportions of CD28- T cells have been shown by several groups of investigators to predict higher risk of adverse outcomes associated with CMV infection4,6,16–22.

We previously showed that CMV-specific CD4+CD27-CD28- T cells function as regulatory T cells (Treg), which may explain their association with adverse outcomes in CMV-infected individuals23–25. These Treg inhibit not only proliferation of autologous peripheral blood mononuclear cells (PBMC) in response to CMV ex vivo stimulation, but also proliferation of PBMC in response to candida, HIV or VZV antigens26. CD4+CD27-CD28- Treg can be expanded and purified from ex vivo CMV-stimulated PBMC or purified from unstimulated circulating PBMC in CMV-seropositive individuals26.

The goal of this study was to determine mechanism/s of action of the CD4+CD27-CD28- Treg, by investigating the phenotypic and functional changes of T cells and antigen presenting cells (APC) after exposure to CD4+CD27-CD28- Treg.

Methods

Study population.

The study used deidentified viably cryopreserved PBMC from CMV-seropositive and seronegative adults. The study was considered exempt by the Colorado Multiple Institution Research Board.

Treg expansion and purification.

Cryopreserved PBMC from CMV-seropositive healthy blood donors were thawed and cultured in the presence of UV-inactivated CMV antigen as previously described26. The antigen, derived from CMV strain AD 169 grown and in human lung fibroblasts tissue culture, was tittered in optimization assays to yield maximum lymphocyte proliferation in the conditions used in this report. The final concentration of antigen in the assays corresponded to approximately 5*104 plaque forming units/ml. Mock-infected control was processed similar to the CMV-antigen but without the viral infection step. After six days of stimulation, PBMC were washed with 2% fetal bovine serum (FBS) in PBS and stained with anti-CD4 APC (BD Biosciences, clone RPA-T4), anti-CD27 PE (BD Biosciences, clone M-T271), anti-CD28-FITC (BD Biosciences, clone CD28.2) for 30 minutes in the dark. After incubation, PBMC were washed with 2% FBS in PBS and CD4+CD27-CD28- cells were separated using an Astrios cell sorter (Beckman Coulter). Cell purity was ≥98%. Sorted CD4+CD27-CD28- cells were cultured with autologous PBMC in the presence of CMV antigen for six days.

Transwell experiments.

Freshly thawed PBMC from CMV-seropositive donors were cultured in 200μl/well growth medium at 90,000 cells/well in the presence of CMV antigen in a 96-well round bottom microplate. 10,000 autologous CD4+CD27-CD28- CMV-induced Treg were added to each well. Parallel cultures were set up in 0.4μm pore transwell plates (Corning) where the PBMC and CMV antigen were added to the bottom chamber and the CMV-induced CD4+CD27-CD28- Treg to the upper chamber of the transwells. Proliferation of CMV-stimulated PBMC was measured by 3H-thymidine incorporation as previously described24,26. Results were reported as stimulation indices (SI).

Proliferation and phenotypic characterization of T cells and APC.

Cryopreserved PBMC from CMV-seropositive donors were thawed and labeled with CellTrace™ Violet (Invitrogen) at 0.5uM final concentration. Labeled PBMC were cultured with CMV antigen in the presence of sorted autologous CD4+CD27-CD28-, which were labeled with anti-CD4 APC, as mentioned above, at a 1 to 10 ratio (CD4+CD27-CD28-:PBMC) at 2*106 cells/mL in growth medium consisting of RPMI 1640 (Sigma) with 10% Human Serum (Corning), 1% L-glutamine (Gibco), 10mM Hepes (Sigma), and 100U/mL Pen/Strep (Gibco). After six days of stimulation, cells were harvested for flowcytometry analysis. T cell proliferation was measured with CellTrace™ Violet dye dilution. For phenotypic characterization, cells were labeled with anti-Caspase-9 FITC (Biolegend) or CellEvent™ Caspase-3/7 Green Detection Reagent for 30 minutes at 37°C and 5% CO2 before washing with PBS and labeling with Zombie Yellow™ viability dye (Biolegend). Next, cells were labeled with anti-CD3 Alexa-Fluor 700 (eBiosciences), anti-CD4 PC 5.5 (Beckman Coulter), anti-HLA-DR APC H7, anti-PDL-1 PE, anti-CTLA-4 PE CF594 (Biolegend), anti-HLA-DR APC H7 (BD Biosciences), anti-CD14 PE Cy7 (BD Biosciences), anti-PDL-1 PE (BD Biosciences), anti-CTLA-4 PE CF594 (BD Biosciences), Annexin V FITC (eBiosciences), 7 Aminoactinomycin D (7AAD; eBiosciences) and/or dump CD3/CD19/CD16/CD56 Alexa Fluor 700 (BD Biosciences). xxx-negative populations were analyzed with a Gallios Flow Cytometer (Beckman Coulter). The gating strategy is shown in the supplementary information.

Blocking experiments.

Freshly thawed PBMC from CMV seropositive and seronegative donors were stained with CellTrace™ Violet and stimulated with CMV antigen in growth medium in the presence of neutralizing mouse monoclonal antibodies (mAb) anti-CTLA-4 (Biolegend; clone L3D10) and/or anti-PD-1 (Biolegend; clone A17788B). After 6 days of incubation, CellEvent™ Caspase-3/7 Green Detection Reagent was added to PBMC cultures 30 min, followed by Zombie Yellow™ viability dye, followed by anti-CD3 Alexa-Fluor 700 (eBiosciences) and anti-CD4 PC 5.5 (Beckman Coulter) for 30 minutes at room temperature. Lastly, cells were washed with Annexin V buffer diluted to 1X (eBiosciences) followed by Annexin V APC (eBiosciences). Cells were analyzed using a Gallios Flow Cytometer.

Statistical analysis.

The distribution of the data was analyzed and log transformation was performed as appropriate to achieve normal distribution. Comparisons were performed by paired or unpaired T test, as appropriate and significant differences were defined by p values < 0.05. The analyses used Prism 8.1.2 software for Mac OS (GraphPad).

Results

Contact requirement for the inhibitory effect of CMV-induced Treg on CMV-specific proliferation of autologous PBMC.

To determine if the effect of CMV-induced Treg on the proliferation of CMV ex vivo stimulated autologous PBMC was mediated by soluble factors or necessitated cell-to-cell contact, we performed transwell experiments. PBMC were incubated with CMV antigen or mock-infected control in 96-well plates and the CMV-induced Treg were added to the upper chamber of transwell plates separated from the lower chamber by a 0.4 μm membrane that prevented passage of cells between chambers, but not of cytokines, chemokines or other soluble factors. In control experiments, the Treg were directly added to the autologous PBMC suspension. Figure 1 shows that CMV-induced Treg inhibited autologous PBMC proliferation measured by 3H-thymidine incorporation only when added to the PBMC suspension, indicating that cell-to-cell contact was necessary for their inhibitory activity.

Figure 1. Contact requirement for the inhibitory effect of CMV-induced Treg.

Figure 1.

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The graphs show individual data points, means and SEM for CMV-stimulated PBMC (CMV) or treated with Treg (CMV+Treg) at a ratio of 1 Treg to 10 PBMC. P values were calculated using paired T-test.

Effect of CMV-induced Treg on T cell phenotypic and proliferative responses to CMV ex vivo stimulation.

To identify the target of the CMV-induced Treg among conventional T cells, we measured ex vivo CMV-stimulated proliferation of CD4+ and CD8+ T cells by flowcytometry. We determined that the addition of CMV-induced Treg decreased proliferation of both CD4+ and CD8+ T cells, but only the effect on CD8+ T cells reached statistical significance (p=0.02; Fig 2).

Figure 2. CMV-induced Treg (Tr) preferentially inhibit proliferation of CD8+ T cells.

Figure 2.

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The graphs show flowcytometry-measured proliferation of CD4+ (left panel) and CD8+ T cells (right panel) in CMV-stimulated (CMV), mock-stimulated (Media) and CMV-stimulated PBMC treated with autologous CMV-induced Treg (CMV+Tr) at 1 Treg: 10 PBMC ratio. CMV-induced Treg were labeled with anti-CD4-APC and the homologous PBMC with anti-CD4-PC 5.5 to allow separation of the two cell populations in the flow analysis. The data obtained with mock-stimulation are included to provide context for the magnitude of the CMV stimulatory effect, but were not included in the statistical anlaysis. The lines link data points for the same donor. There was a significant difference in CD8+ T cell proliferation between CMV and CMV+Tr condition (p=0.02; paired T test).

To gain further insight into the mechanism of action of CMV-induced Treg, we studied the phenotypic profile of T cells stimulated ex vivo with CMV antigen and co-cultured with autologous CMV-induced Treg by comparison with untreated controls. We focused on activation and regulatory markers, including HLA-DR and PD-L1; as well as apoptosis, including expression of activated Casp-3 and binding of Annexin-V. After 6 days of CMV ex vivo stimulation, CD4+ and CD8+ T cells treated with CMV-induced Treg had significantly decreased proportions of cells expressing PD-L1 and/or HLA-DR and increased proportions of cells binding annexin V and/or exhibiting bright Casp-3 expression compared with untreated controls (Fig 3).

Figure 3. Phenotypic changes of CMV-stimulated T cells after CMV-induced Treg exogenous supplementation.

Figure 3.

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Graphs show individual data points, means and SEM of CD4+ (upper panels) and CD8+ (lower panels) in CMV-stimulated PBMC cultures treated with autologous CMV-induced Treg (CMV+Treg) at 1 Treg: 10 PBMC ratio or not (CMV). CMV-induced Treg were labeled with anti-CD4 APC and the homologous PBMC with anti-CD4 PC 5.5 to allow separation of the two cell populations in the flow analysis. P values indicated on the graphs were calculated using paired T-tests. Abbreviations: AnV=Annexin V; Casp3=Caspase 3

Effect of CMV-induced Treg on APC phenotypic characteristics after ex vivo stimulation with CMV antigen.

The effect of CMV-induced Treg on autologous CMV ex vivo-stimulated APC was investigated by Annexin-V binding, 7-AAD uptake, Casp-3 and Casp-9 activation, and expression of PD-L1 and CTLA-4 on lineage negative HLA-DR+CD14+ monocytes and HLADR+CD14- dendritic cells (DC). CMV-induced Treg significantly increased the CTLA-4 expression and Annexin-V binding on DC (Fig 4). 7-AAD uptake by DC increased in 3 out of 4 samples tested, but failed to reach statistical significance. There were no appreciable changes in the monocyte phenotypic characteristics or apoptosis markers associated with CMV-induced Treg treatment (not depicted).

Figure 4. Phenotypic changes of CMV-stimulated APC after CMV-induced Treg exogenous supplementation.

Figure 4.

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Graphs show individual data points, means and SEM of HLADR+CD14- DC in CMV-stimulated PBMC cultures treated with autologous CMV-induced Treg (CMV+Treg) or not (CMV). P values indicated on the graphs were calculated using paired T-tests. Abbreviations: AnV=Annexin V; 7AAD=7 Aminoactinomycin D

Ex vivo modulation of Treg inhibitory activity with pathway blocking agents.

To determine if the increased expression of CTLA-4 on DC mediated the inhibitory effect of Treg, we compared CD4+ and CD8+ T cell responses to CMV-ex vivo stimulation in the presence of anti-CTLA-4 blocking mAb or the presence of anti-PD-1 blocking mAb, which we previously showed to mitigate the Treg inhibitory effect on T cell proliferation24. In these experiments, we relied on circulating Treg to downmodulate DC function based on our previous observations that circulating Treg in CMV-seropositive individuals share the properties of CMV-induced Treg26. Figure 5 shows that anti-PD-1 significantly increased the proliferation of CD8+ T cells in response to CMV ex vivo stimulation (p=0.04) and to a lesser extent that of CD4+ T cells (nonsignificant). In contrast, the anti-CTLA-4 treatment marginally decreased the CD4+ T cell proliferation (p=0.08) and had no effect on CD8+ T cell proliferation. The overall effect on total CD3+ T cell proliferation consisted of significant increases in response to anti-PD-1 treatment (p=0.03) and decreases in response to anti-CTLA-4 (p=0.02). We further investigated the effect of anti-PD-1 on T cell apoptosis and determined that it was associated with significant decreases in CD4+ and CD8+ apoptosis measured by Caspase-3 activation and Annexin-V binding, particularly on the Violetlo proliferating CD4+ and CD8+ T cells (Fig 6).

Figure 5. Anti-PD-1 (aPD-1), but not anti-CTLA-4 (aCTLA-4) blocking antibodies increase CMV-stimulated T cell proliferation.

Figure 5.

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Graphs show individual data points, means and SEM for the stimulation conditions indicated on the x axis. Asterisks indicate significant differences (p<0.05) of CMV-stimulated controls with aPD-1 or aCTLA-4 treated cultures.

Figure 6. Anti-PD-1 (aPD-1), but not anti-CTLA-4 (aCTLA-4) blocking antibodies decrease CMV-stimulated T cell apoptosis.

Figure 6.

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Graphs show individual data points, means and SEM for the stimulation conditions indicated on the x axis. Asterisks indicate significant differences (* 0.01<p <0.05; ** p <0.01) of CMV-stimulated controls with aPD-1 or aCTLA-4 treated cultures. Abbreviations: AnV=Annexin V; Cas=Caspase 3

Discussion

Our results show that CMV-induced Treg predominantly affected CMV-specific CD8+ T cells, which displayed the most significant changes in co-cultures of Treg and CMV-stimulated autologous PBMC. Compared with CD4+, proliferation and activation of CD8+ T cells was more vigorously inhibited by Tregs. Both T cell subsets displayed increased markers of apoptosis after exposure to Treg. We hypothesized that CD8+ T cells might be more susceptible than CD4+ T cells to the Treg effect due to their dependence on trans-stimulation of IL2 secreted by CD4+ T cells, while CD4+ T cells could use IL2 in cis, leaving them less susceptible to depletion of IL2 by Treg27,28. However, in experiments in which we added rhIL2 to the Treg-treated PBMC during CMV stimulation, CD4+ and CD8+ T cells demonstrated similar increases in proliferation (data not shown). The requirement of cell contact between Treg and PBMC also argues against IL2 depletion being a major mediator of the Treg effect. Although the predilection of the Treg effect on CD8+ T cells makes teleological sense, because the CD8+ T cells are cytotoxic and more destructive than their CD4+ counterparts, the mechanism by which Treg specifically target CD8+ T cells remains to be elucidated.

Consistent with the predominant effect of Treg on CD8+ T cells, addition of anti-PD-1 to ex vivo CMV-stimulated PBMC significantly restored proliferation of CD8+ T cells and had a much smaller, statistically nonsignificant effect on CD4+ T cells. CD8+ T cells represented a substantial contingent of the proliferating T cells in CMV-stimulated PBMC, such that the effect of Treg and of anti-PD-1 on CD8+ T cells translated into an overall effect on CD3+ T cells.

We further showed that anti-PD-1 treatment was associated with a reduction in apoptosis of CMV-specific T cells, consistent with restoration of their ability to proliferate.

In addition to PD-1 blockade, we showed in our previous studies that anti-TGFβ neutralizing monoclonal antibodies and the granzyme B inhibitor II attenuated the activity of CMV-induced Treg. However, here we found that Treg needed cell-to-cell contact for their function, which may seem contradictory to the ability to modulate the Treg effect with anti-TGFβ or granzyme inhibitors. A model that reconciles that two sets of observations is that the anti-TGFβ neutralizing antibody acted by inhibiting the recruitment of new Treg from the autologous PBMC rather than blocking the activity of pre-expanded Treg. The granzyme B II inhibitor is also an inhibitor of various caspases. Here we showed that increased apoptosis of T cells and APC is one of the effects of Treg on autologous PBMC. It is conceivable that the granzyme B inhibitor II rescued cells from apoptosis and allowed them to proliferate under CMV ex vivo stimulatory conditions counterbalancing the Treg mechanism of action.

One of the Treg mechanisms of action is induction of tolerogenic APC29–31. In our co-culture experiments, CMV-induced Treg had a detectable effect on DC and no appreciable effect on monocytes. DC in Treg-treated preparations showed increased apoptosis and increased expression of CTLA-4, which is an inhibitory ligand of CD28. CTLA-4, like PD-1, has been targeted by anti-tumor immunologic checkpoint blockade therapy and may be considered in the combat of chronic infections32. However, CTLA-4 blocking experiments did not mitigate the effect of Treg on CMV-specific proliferating CD4+ and CD8+ T cells. Paradoxically, the addition of anti-CTLA-4 blocking mAb decreased T cell proliferation. This observation does not exclude an in vivo tolerogenic effect of CMV infection on DC. Additional studies are needed to compare DC function from CMV-infected and uninfected individuals.

Our study was limited by the small number of samples and the limited phenotypic markers investigated. In addition, in vitro experiments may incompletely represent in vivo phenomena.

Treg human studies have focused mostly on cancer and autoimmune disorders. Here we showed that Treg associated with CMV infection decrease CMV-stimulated T cell proliferation and activation and increase apoptosis, which can be reversed by blocking the PD-1 pathway. These data may be particularly relevant to CMV-seropositive older adults, who have increased CD4+CD28- and CD8+CD28- T cell numbers positively correlated with immune-suppression4,6,21 and to CMV-seropositive transplant recipients, who also display increased Treg and in whom CMV-seropositivity is associated with superinfections33,34.

Supplementary Material

1

Acknowledgments:

This study was partially funded by R21 AI073121.

Footnotes

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Conflict of Interest: AW receives research grants from Merck & Co. Inc..

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Supplementary Materials

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NIHMS1595803-supplement-1.docx (1.7MB, docx)

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