Short Communication: Human Bone Marrow Stromal Cells Exhibit Immunosuppressive Effects on Human T Lymphotropic Virus Type 1 T Lymphocyte from Infected Individuals

Abstract

Human multipotent mesenchymal stromal cells (MSCs) display immunoregulatory functions that can modulate innate and adaptive cellular immune responses. The suppressive and immunomodulatory activities of MSCs occur through the action of soluble factors that are constitutively produced and released by these cells or, alternatively, after MSC induction by stimuli of inflammatory microenvironments. However, to date the contribution of MSCs in the inflammatory microenvironment resulting from viral infection is unknown. In our study, we evaluated the MSC immunosuppressive effect on human T lymphotropic virus type 1 (HTLV-1) infected T lymphocytes. To evaluate if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of lymphocytes obtained from HTLV-1 infected and healthy individuals cocultured in the presence of MSCs. It was observed that the lymphoproliferative inhibition by MSCs on infected lymphocytes was similar compared to the cells obtained from healthy individuals. In addition, this suppressive effect was related to a significant increase of indoleamine-2,3-dioxygenase and prostaglandin E2 gene expression (p ≤ .05). Furthermore, the HTLV-1 pol gene was less expressed after coculturing with MSCs, suggesting that the MSC immunoregulation can have effective suppression on HTLV-1 infected T cells. In conclusion, this study suggests that MSCs could be involved in the immunomodulation of the HTLV-1 infected T lymphocytes.

The ability of human multipotent mesenchymal stromal cells (MSCs) to suppress T lymphocyte proliferation and modulate the immune response has been explored for therapeutic approaches in the treatment of inflammatory, autoimmune, and graft-versus-host disease.¹ The precise mechanisms by which MSCs modulate the immune response have not yet been fully elucidated; however, adhesion molecule expression and the secretion of soluble factors are considered important mediators for MSC immunosuppressive effects.² Another important factor is the environmental condition in which the MSC is exposed. The therapeutic effects of MSCs depend largely on the inflammatory environment and this fact should be considered during application of cell therapy.³ For this reason, there is a growing interest in better understanding the immune properties of MSCs during physiological and pathological conditions.

In the natural course of human T lymphotropic virus type 1 (HTLV-1) infection, a systemic inflammatory response related to the release of different pro-inflammatory factors can be observed. In regards to this, the coculture of murine bone marrow stromal cells with T cells derived from adult T cell leukemia (ATL) cell lines supports the cell growth and survival, although downregulating the transcriptional and translational levels of HTLV-1 Tax.⁴ However, the exact mechanism of interaction between stromal cells and HTLV-1-infected T cells is unknown.

In this study we evaluated the immunomodulatory properties of MSCs on T lymphocytes isolated from HTLV-1-infected individuals. Initially, bone marrow-derived MSCs were obtained from healthy donors, and cell morphology, surface markers, and differentiation potential in mesodermal lineage cells were evaluated by different methods (microscopy, flow-cytometry analysis, induction with specific mediums, and staining). MSCs showed adherence to plastic surface and spindle-like morphology when maintained in standard culture conditions. In addition, they expressed CD73 (90.78% ± 7.29%), CD90 (98.22% ± 0.59%), and CD105 (90.65% ± 6.14%) and lacked expression of CD34 (0.35% ± 0.25%), CD31 (0.90% ± 0.42%), CD45 (1.13% ± 1.24%), CD14 (0.99% ± 1.38%), and human leukocyte antigen HLA-DR. Furthermore, they had the potential to differentiate into osteoblasts and adipocytes in vitro (Fig. 1I–III).

FIG. 1.

Morphological characterization and cellular plasticity of MSC isolated healthy donors. MSCs cultured on standard medium stained with hematoxylin and eosin (AI); osteogenic differentiation shows the mineralization nodules analyzed by von Kossa method (AII) and adipogenic differentiation demonstrated by Sudan II/Scarlet staining of lipid vacuoles (AIII). The scale bars represent 100 μm. Proliferation percentages of T-CD4⁺ (B) and T-CD8⁺ (C) by CFSE detection using flow cytometry. White bars represent positive control group of peripheral blood lymphocytes CFSE labeled and stimulated by PHA; Bars in gray represent the inhibitory activity of healthy MSCs in coculture with allogeneic lymphocytes stimulated by PHA in ratio (MSCs:PBMCs = 1:5); Bars in black represent the inhibitory activity of healthy MSCs in cocultures with allogeneic lymphocytes stimulated by PHA in ratio (MSCs:PBMCs = 1:20). Data are showed as mean ± SEM of triplicate assays. CT = control subjects (n = 05); HAC = asymptomatic HTLV-1⁺ individuals (n = 08); HAM = HTLV-1⁺ individuals with HAM/TSP (n = 10). Gene expression by real time of IDO (D) and PGE2 (E). The black bars represent MSC; the blank bars represent PBMCs of control subjects (CT); Bars in gray represent the PBMC of HAC, and dark gray bars represent PBMCs of HAM/TSP. The (−) signal indicates PBMC grown in the absence of MSCs, and the (+) signal indicates PBMC cocultured with MSCs. HTLV-1 pol gene expression by real-time PCR (F, G). Data are reported as ERU. The results are represented as the mean ± SEM of duplicate assays. Control subjects (n = 5), HAC (n = 07); HAM/TSP (n = 7). For the realization of the real-time PCR expression analysis, we performed RNA extraction using TRIzol^® reagent, followed by RT-PCR and mRNA gene expression by TaqMan^® real-time PCR. Real-time PCR was performed in three different cell populations: (i) MSCs, cultured during 5 days and harvested using 1 × trypsin; (ii) cell suspension of PBMCs after 5 days of culture; and (iii) total population of PBMCs in coculture for 5 days with MSCs. Detection of HTLV-1 p19 antigen by ELISA (H). Quantification by ELISA of the p19 antigen present in the cell culture supernatant. White bars represent MT-2 cells, and bars in black represent the MT-2 cell coculture with MSCs. The asterisks denote significant differences (*p ≤ .05, **p ≤ .005) using one-tail Mann–Whitney T-test. CT, control subjects; CFSE, carboxyfluorescein succinimidyl ester; ELISA, enzyme-linked immunosorbent assay; ERU, expression relative units; HAC, HTLV-1 asymptomatic carriers; HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesis; IDO, indoleamine 2,3-dioxygenase; MSC, mesenchymal stromal cell; PCR, polymersae chain reaction; PGE2, prostaglandin E2; PHA, phytohemagglutinin; RT-PCR, reverse transcription-PCR; SEM, standard error of the mean. Color images are available online.

To determine if MSCs are able to inhibit the proliferation of HTLV-1-infected lymphocytes stimulated with phytohemagglutinin (PHA), we performed a lymphoproliferative assay using PBMCs obtained from HTLV-1-infected individuals cocultured with MSCs isolated from healthy donors. The HTLV-1-infected group consisted of eight healthy HTLV-1 asymptomatic carriers (HAC) (three females and five males; mean age, 41.5 years) and ten HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) individuals (six females and four males; mean age, 53.1 years). The healthy control was composed of five randomly selected volunteer blood donors (three females; two males; mean age, 38 years). All recruited individuals signed a written informed consent (Ethical approval by the Faculty of Medicine of Ribeirão Preto, USP, #12462/2010).

According to Figure 1, MSCs significantly inhibited in vitro CD4⁺ and CD8⁺ T cell proliferation in a dose‐dependent manner (p < .05) (Fig. 1B, C). In addition, we observed that inhibitory effects occurred in both lymphocytes isolated from HTLV-1 infected individuals and healthy controls. However, when we compared the relative inhibitory effects among the three groups of samples (asymptomatic HTLV-1 infected individuals, HAM/TSP patients, and control subjects), no significant difference was observed.

To understand if the MSCs ability to inhibit HTLV-1⁺ T cell proliferation could be related to release of soluble factors, we investigated with real-time polymerase chain reaction (PCR) the expression profile of genes related to MSC immunoregulation like indoleamine-2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). IDO activity causes depletion in the synthesis of tryptophan and quinurenin, inhibiting the growth and function of immune cells due to lack of nutrients or direct toxicity in their catabolism. The immunosuppressive effects of PGE2 are related to the modulation of the secretion profile of cytokines especially interleukin (IL)-2 and interferon (IFN)-γ, altering from an inflammatory to anti-inflammatory environment. We observed that the mRNA expression of the molecules IDO and PGE2 was upregulated (p ≤ .05) in MSCs cocultured with peripheral blood mononuclear cells (PBMC), indicating that T cell suppression mechanisms used by MSCs also may occur during HTLV-1 infection. However, no significant differences in IDO/PGE2 expression were observed between MSCs cocultured with control PBMC from blood donors or HTLV-1-infected individuals (Fig. 1D, E). The gene expression of the LGALS1, vascular cell adhesion protein 1 (VCAM-1), and IL-6 was similar in MSCs cultured alone and cocultured with PBMC (Appendix Fig. A1).

We also investigated the role of MSCs in the modulation of HTLV-1 gene expression. The real-time PCR quantification showed a lower expression of HTLV-1 pol gene (7.3-fold) in HTLV-1⁺ PBMC cocultured with MSCs compared to PBMCs cultured in the absence of MSCs (p = .0128) (Fig. 1F). This significant decrease was also observed in samples from HTLV-1⁺ HAM/TSP individuals cocultured with MSCs (p = .0325) (Fig. 1G). In addition, the expression of HTLV-1 p19 protein into supernatant from HTLV-1⁺ PBMC cocultured with MSCs was 1.8-fold lower compared to PBMC cocultured without MSC, without statistical significance (data not shown). However, when we evaluated the supernatant from HTLV-1 infected cell line MT-2, cocultured with MSCs, a significant reduction of HTLV-1 p19 protein was observed (Fig. 1H). Finally, we evaluated the ability of MSCs to induce subpopulations of regulatory cells, but no significant differences were found in the percentages of recovered regulatory cell subpopulations (CD4⁺CD25^hiFOXP3⁺, CD4⁺HLA‐G⁺, CD8⁺HLA‐G⁺, and CD3⁺) (p > .05) (Table 1).

Table 1.

Subpopulation Cell Analyses After Mesenchymal Stromal Cell Coculture Assay with Lymphocytes

Cell population	CT	CT + MSC	HAC	HAC + MSC	HAM/TSP	HAM/TSP + MSC	p
HLAG⁺CD4⁺CD3⁺	7.3 ± 5.6	12.8 ± 11.5	8.9 ± 5.3	7.8 ± 5.5	10.6 ± 8.8	6.1 ± 7.1	.44
HLAG⁺CD8⁺CD3⁺	8.0 ± 4.7	13.3 ± 14.6	9.3 ± 5.8	5.8 ± 1.9	5.8 ± 3.0	4.4 ± 4.6	.94
CD4⁺CD25high⁺FoxP3⁺	18.4 ± 25.9	6.1 ± 5.0	206 ± 20.2	12. ± 10.6	15.8 ± 14.2	10.5 ± 8.4	.56

CT, control subjects; HAC, HTLV-1 asymptomatic carriers; HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesis; MSC, mesenchymal stromal cell.

The MSC coculture with PBMC isolated from HTLV-1-infected individuals resulted in a significant inhibition of HTLV-1 pol gene. Similar data were described by Kinpara⁵ who observed a significant decrease in gag expression of HTLV-1-infected T lymphocytes after coculture with mouse stromal cells. The authors suggest that viral suppression occurred in response to the production of IFN-α and IFN-β by the stromal cells.⁵ Differently to Kinpara,⁵ we used human stromal cells from bone marrow of healthy individuals, and our results indicate that viral suppression may have occurred due to the immunosuppressive effect on alloreactive T lymphocytes by MSCs, since the proliferation of CD4⁺ T and CD8⁺ T lymphocyte stimulated PHA isolated from HTLV-1 individuals was significantly lower when cocultured with MSCs.

Previous studies have investigated the MSCs immunosuppressive capacity during Epstein–Barr virus, adenovirus, and cytomegalovirus (HCMV) infections.^6,7 These studies questioned if MSC treatment by infusion in immunocompromised individuals could suppress innate immune responses to pathogens and result in a higher susceptibility to infectious complications. Indeed, the MSCs have differential immunomodulatory effects on viral infection, not being able to prevent the proliferation of virus-specific cytotoxic T lymphocytes (CTLs), but allowing a production of virus-induced IFN-γ.⁷ Therefore, the use of MSCs in allogeneic stem cell transplantation does not increase the severity of the infections and particularly the risk of viral reactivation especially in the case of HCMV.⁶

According to our results, the mechanisms for T lymphocytic suppression used by the MSCs in HTLV-1-infection are similar to those occurring under physiological conditions. The gene expression of immunoregulatory molecules like IDO and PGE2 was significantly increased in MSCs cocultured with PBMCs, regardless of virus infection or the clinical status (healthy donors, HAC, or HAM/TSP). The relationship between MSC immunosuppressive effect and the expression of molecules, including TGF-β1, IDO, PGE2, VCAM-1, and nitric oxide (NO), has been described in the literature.⁸ To confirm the exact function of the IDO and PGE2 in the HTLV-1-infected T cell proliferation, future studies must be performed using shRNA inhibition assays. However, in our case we could not collect new samples to complete this analysis due to lost patient follow-up and ethics requirements.

The MSC immunomodulation properties also depend on external signals. For example, MSCs treated in vitro with specific molecular patterns associated with pathogens (PAMP) may become anti- or pro-inflammatory dependent on PAMP with which they interact.² This differentiation behavior of MSCs is due to the fact that these cells express toll-like receptors 3 and 4 (TLR3 and TLR4), which recognize double-stranded RNA, single-stranded RNA, and some bacterial products. The interaction of TLR3 and TLR4 with a pathogen is associated with inhibition of MSC activity in suppressing CD4⁺ T lymphocyte proliferation, without influencing the immunophenotype or cellular plasticity of MSCs.⁹ Other studies demonstrate that the TLR3 and TLR4 stimulation by PAMP increases the immunosuppressive properties of MSCs through IFN-β and IDO-1 expression.¹⁰ According to these findings, it was expected that the MSC activity in infectious microenvironment, such as in the presence of HTLV-1-infected cells, could be differentiated. However, in this study, we found no specific effects in relation to intensity of immunosuppression, since MSCs cocultured with lymphocytes obtained from HTLV-1-infected individuals and lymphocytes isolated from healthy individuals had similar immunosuppressive effects. Nevertheless, the immunomodulatory effect of the MSCs on HTLV-1 infected T lymphocytes may serve as a promising tool for understanding the pathogenetic mechanisms of HTLV-1 induced malignant proliferative diseases like ATL/lymphoma.

Footnotes

Acknowledgments

The authors are grateful to the staff and patients of the Neurology Department of the University Hospital. The authors also thank Camila C.B.O. Menezes for flow cytometry analysis, Sandra Navarro Bresciani for the artwork, and Andy Alastair Cumming for carefully reading the article. Funding: this work was supported by CNPq Universal (process no., 477126/2009-0). Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)—Center for Cell-based Therapy (CTC) (process no. 2013/08135-2), the Conselho Nacional do Desenvolvimento Científico e Tecnológico, Brazil (INCTC-465539/2014-9), and Fundação Hemocentro de Ribeirão Preto (FUNDHERP).

Author Contributions

E.S.R., designed research studies, conducted experiments, acquired and analyzed data, and wrote the article; O.M.T., HTLV-1 patients' medical assistance; M.D.M., M.D.O., and M.T.P., cell culture assistance; P.V.B.P., acquired and analyzed flow cytometry data; S.N.S., analyzed data and wrote the article; G.L.V.O. and K.C.R.M., cocultured assay assistance; and D.T.C. and S.K., oversaw experimental design, data analysis and interpretation, and edited the article. All authors read and approved the final article.

Author Disclosure Statement

No competing financial interests exist.

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