In this issue of AIDS, Zhang et al.  report about safety and immunological responses of therapy with mesenchymal stem cells (MSCs), derived from human umbilical cord or more specifically Wharton's jelly, in HIV-infected nonimmune responder (NIR) individuals. The NIRs respond to highly active antiretroviral therapy (HAART) and effectively suppress HIV replication, but do not show any improvement in their immune status, as measured by increase in CD4+ T-cell counts. The authors conducted a prospective and open-label controlled pilot clinical trial. They made 3-monthly intravenous MSC transfusions in seven NIRs, whereas six NIRs received saline as controls. All the participants remained on HAART during the study and a 12-month follow-up period. The MSC recipients tolerated the therapy well and showed neither any adverse clinical effect nor increase in viral burden. More importantly, the MSC recipients showed a significant increase in their naive and central memory CD4+ T-cell counts compared with the control group. The therapy also restored their ability to produce interleukin (IL)-2 and IFN-γ in response to HIV antigens, and down-regulated signs of immune activation, immune exhaustion, and inflammation. This study has provided a new glimpse of hope for the HIV-infected NIRs. It is noteworthy that more than 20% HAART-treated HIV-infected persons exhibit the NIR phenotype, and are at increased risk for opportunistic infections, cancer and reduced life expectancy .
Mesenchymal stem cells are multipotent self-renewing precursor cells that can differentiate, under appropriate stimuli, into cells of the mesodermal origin, that is, osteoblasts, chondrocytes, muscles, adipocytes, and so on. They can also trans-differentiate into cells of ectodermal and endodermal origins such as neurons, hepatocytes, pancreatic islet cells, and so on [3,4]. They secrete many trophic, angiogenic, and mitogenic factors, which impart them tissue regenerative capacity. MSC-based cell therapies are under intensive investigation for bone repair, acute kidney injury, neuronal injury, myocardial infarction, intervertebral disc repair, and neurodegenerative diseases like Alzheimer's disease, and so on. Furthermore, they also possess anti-inflammatory and immunosuppressive properties [5,6]. They secrete monocyte chemoattractant protein 1, attract and kill T cells via Fas Ligand and TNF-related apoptosis-inducing ligand. They also secrete several other immune modulating substances like prostaglandin E (PGE)-2, human leukocyte antigen-G5, IL-10, nitric oxide, heme oxygenase 1, leukemia inhibitory factor, transforming growth factor (TGF)-β1, IL-6, indoleamine 2,3-dioxygenase, and so on, which enable them to inhibit natural killer (NK) and T-cell responses, and promote expansion of FoxP3-positive Treg cells. The cells also inhibit/delay maturation of dendritic cells and promote their differentiation into tolerogenic phenotype. Furthermore, they have also been shown to promote macrophage differentiation into type 2 anti-inflammatory phenotype [5,6]. For these properties, MSCs are also being investigated for cell therapies in chronic inflammatory and autoimmune diseases like multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, graft versus host disease (GvHD), and so on [3,4,7,8]. Genetically modified MSCs are also being tested to deliver anticancer drugs/molecules in cancer patients .
Because of their immunosuppressive properties, the use of MSCs in HIV-infected patients had never been considered in the past. In fact, it is the hematopoietic stem cells (HSCs) that have been/are under intense investigation for their use, after appropriate genetic modifications, for providing ‘functional cure’ from HIV infection [9–11]. Impetus to these investigations was provided by the so-called ‘Berlin patient’. The patient, a 42-year-old HIV-infected German who also suffered from acute lymphoblastic leukaemia (AML), was transfused with allogeneic HSC with the mutant CCR5 after total body irradiation. In 5 years’ follow-up, and to this day, the patient has controlled the infection and the viral RNA has remained undetectable in his plasma despite receiving no antiretroviral drugs. This is the only case of ‘functionally’ cured HIV-infected patient: ‘functionally’ since the patient has not got rid of the virus from his body [12,13]. It is believed that the patient controlled the infection because of the mutant CCR5. It is supported by the fact that the individuals homozygous for the delta 32 CCR5 are resistant to the infection and the individuals heterozygous for this mutation have better prognosis . However, it is noteworthy that the patient also underwent various chemotherapies for GvHD and AML relapses. How these therapies affected the HIV infection remains unknown. Despite these caveats, such ‘functional’ cures are actively being investigated for HIV-infected individuals with lymphomas. In these attempts, CCR5 in the donor HSC is disrupted by different approaches, for example, gene-specific designer zinc finger nucleases, ribozymes, shRNA, siRNAs using different methods like electroporation, lentiviral or adenoviral vectors, and so on [10,11]. In addition, HSCs expressing different anti-HIV cellular factors (TRIM5-α, APOBEC-3G/C) are also being considered. A serious limitation of using allogeneic HSCs in HIV-infected or any other patient is their strong immunogenicity, and ability to induce GvHD. The recipient has to undergo immunosuppressive regimens. Unlike HSCs, allogeneic MSCs are considered nonimmunogenic, and no immunosuppression of the recipient is required. In future, when appropriate protocols are developed for using MSCs for inhibiting GvHD, co-administrations of allogeneic MSCs and HSCs without the need for immunosuppression may be possible.
The exact mechanism how allogeneic MSCs reconstitute the immune system in HIV-infected recipients remains unknown. They may have done so through attenuating inflammation and promoting lymphopoiesis. In addition to secreting anti-inflammatory substances (PGE2, TGF-β1, etc.), MSCs have been shown to increase intestinal barrier function . Thus the transfused cells are likely to reduce microbial translocation in HIV-infected patients. The translocation is recognized as a cause of chronic systemic immune activation in these patients. In addition, MSCs are known to provide structural and functional support (niche) for HSCs in bone marrow and T-cell precursors in the thymus. They produce CXCL-12 and IL-7 , the latter being an essential cytokine for lymphopoiesis, T-cell growth, and survival. Therefore, MSCs are likely to ameliorate diminished regenerative capacity of HSCs in HIV-infected individuals. Thus, immune reconstitution and increased numbers of CD4+ T cells reported by Zhang et al. were not entirely unanticipated.
Zhang et al. used MSCs from Wharton jelly. However, there exist many other sources of MSCs in adult and fetal tissues: bone marrow, adipose tissue, peripheral blood, cord blood, placenta, menstrual blood, and so on. The MSCs obtained from different sources are likely to show differences in their phenotypic and functional characteristics [3,7,8]. It would be highly desirable to determine which of them have better abilities to reconstitute the immune system. Furthermore, since a large number of MSCs are needed for transfusions, it necessitates their in-vitro expansion. It has been well documented that different culture protocols and treatments (e.g. TLR agonists) modulate anti-inflammatory, tissue regenerative, and homing properties of MSCs in different ways [9,16]. Researchers need to standardize these protocols so that the expanded MSCs retain their immune reconstituting and anti-inflammatory properties.
Old dogma that transfused MSCs preferentially migrate to the site of tissue injury and differentiate into and/or fuse with tissue cells and restrain damage is no longer supported by current research. Several studies, based upon the recovery of fluorescent tags, have shown that only a very small fraction of the transfused MSCs migrates to, and engraft in, the damaged tissues in the body . In fact, a recent study, based upon the recovery of live cells, has demonstrated that i.v. transfused MSCs are trapped in the lungs, and do not migrate to any other tissue . The cells undergo apoptosis within 24 h, and are engulfed by macrophages/dendritic cells. The scavenger cells may carry the tag to other sites in the body. The authors propose that massive engulfment of apoptotic cells turns macrophages and dendritic cell tolerogenic. However, it does not explain how the transfused MSCs mediate immune reconstitution described by the Zhang et al. study. They certainly release cytokines and trophic factors, which act in a paracrine manner to enhance proliferation and tissue repair in endogenous cells. The conditioned medium from in-vitro MSC cultures exerts MSC-like biological effects. More importantly, new data are emerging to show an important role of the MSC-released microvesicles in mediating various biological effects of the transfused cells [19,20]. In this connection, it has been shown that MSCs release these microvesicles, which express surface receptors characteristic of the parent cells, and are preferentially enriched for certain proteins, lipids, transcription factors, cytokines, mRNAs, miRNAs, and so on [20,21]. The microvesicles migrate and fuse with, and transfer information horizontally to endogenous cells, and reprogram them through epigenetic changes to proliferate, differentiate and repair the damage. The MSC-released microvesicles have been shown to mimic the biological effects of the transfused MSCs. Treatment of microvesicles with RNAse abrogates their MSC-like effects. In this connection, Biancone et al. have made a very provocative proposition of an ongoing cross-talk between differentiated tissue cells and their precursor progenitor cells: cells in an injured tissue release microvesicles that migrate and fuse with progenitor stem cells, and induce the progenitor cells to release their microvesicles, which in turn migrate to the injured tissue and reprogram them to proliferate. The progenitors also undergo proliferation and recruitment to the injured tissues. This microvesicle-mediated cross-talk between injured tissues and progenitor stem cells seems to play an important role in homeostasis and repair of different tissues in the body.
As demonstrated by Zhang et al. and shown by others , the transfused MSCs are generally safe for the recipients. However, they have been reported to undergo undesirable differentiation leading to accumulation of osteocytes in myocardium, adipocytes in kidney, and fibroblasts in lungs . There is also evidence suggesting differentiation of MSCs into immunosuppressive tumor-associated fibroblasts . Moreover, immunogenicity of allogeneic MSCs has also been reported in certain situations [24,25]. Therefore, the transfused patients need to be carefully followed for such adverse events. The infusions of MSC-derived microvesicles or adipose tissue-derived MSC from the same individual may be safer options. Both of them can be engineered with the desired homing properties.
In brief, the study by Zhang et al. has made a seminal observation and underpinned immune enhancing effects of MSCs in NIRs. Like any other novel study, it has also raised several questions. We anticipate that more studies will be forthcoming in the near future to answer these questions and explore the effects of MSCs from divergent sources on immune functions in HIV-infected patients as well as in other diseases with compromised immune system.
Note added to the proofs: other than the first functionally cured ‘Berlin patient’ mentioned above, a second case was reported in the 2013 annual Conference on Retroviruses and Opportunistic Infections (CROI) that was held in Atlanta (USA) on 3–6 March 2013. This is the case of a premature baby born to an HIV-infected mother, who was unaware of being infected with the virus. The physicians administered antiretroviral therapy (ART) in unusually high doses to the HIV-positive baby within 30 h after birth. The baby (now two and a half years old) seems to be cured, as she has no detectable viral RNA in her plasma despite not receiving any ART for the last 10 months. The results suggest that infected persons could be cured if the ART is administered before the virus is able to establish its ‘reservoirs’ in the body.
The authors thank the colleagues who exchanged their views and/or took part in discussions on the subject.
Conflicts of interest
The authors declare no competing interests.
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