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Vγ9Vδ2 γδ T cells have effector potential against several cancers and infectious agents. Whether these cells control HIV replication was tested in humanized mouse model.
NOD SCID gamma mice engrafted with human peripheral blood mononuclear cells (PBMCs) and infected with HIVBAL were treated with zoledronate-expanded Vγ9Vδ2 T cells either alone or in combination with an anti-HIV envelope antibody b12.
Severe depletion of CD4+CCR5+ T cells was observed in PBMCs of all infected mice. HIV plasma p24 levels were comparable in all infected groups with no decrease in plasma viremia achieved by adoptive transfer of cells.
Autologous transfer of ex vivo expanded Vγ9Vδ2 T cells may not be a successful treatment choice for controlling HIV replication in vivo.
The phenotype of majority of peripheral blood γ/δ T cells is Vγ9Vδ2 and cytotoxicity of these cells is well demonstrated against solid tumors, melanoma, myeloma and lymphoma [1,2]. Among infectious diseases, expansion of this subset of T cells is observed in Mycobacterium tuberculosis, malaria and bacterial infections [3,4]. Adoptive transfer of ex vivo aminobisphonate-expanded Vγ9Vδ2 T cells is being tested for therapeutic potential against metastatic solid tumors , where such treatment showed some additive effect when given alongside previously ineffective cancer therapy. Recently, a role for pamidronate expanded Vγ9Vδ2 T cells against influenza virus was shown . These cells effectively lysed influenza virus infected cell lines through the Fas-FasL and perforin-granzyme B pathways. In a humanized mouse model, cells expanded by aminobisphosphonate pamidronate reduced disease severity and mortality caused by seasonal and avian influenza virus .
While it is confirmed that there is significant depletion in both numbers and function of Vγ9Vδ2 T cells in HIV infected individuals [8,9], whether this subset has an anti-HIV effect remains to be established. Natural history studies have shown a correlation between Vδ2 T cell frequency and lower vRNA  and reconstituted Vδ2 T-cell numbers are observed in elite controllers of HIV . Cytolytic capacity of these cells against HIV-infected cells has been shown using in vitro systems [12,13]. The mucosal γδ T cells were expanded in rhesus macaques that were protected in a challenge study indicating their role in protection , however, these mucosal cells are primarily of Vδ1 subtype. In sooty mangabeys, the natural hosts of SIV, dysfunction of Vγ9Vδ2 T cells is not observed  suggesting that the dysfunction observed in pathogenic HIV infection may play role in disease progression.
Use of humanized mouse model can be an efficient way to observe direct effects of such interventions like adoptive cell transfers on HIV pathogenesis. 4- to 6-week-old NOD SCID gamma (NSG) mice were used for engraftment of human peripheral blood mononuclear cells (PBMCs). These mice lack the IL-2 receptor γ chain and are defective in cytokine signaling, leading to successful engraftment of human cells. All protocols described here were approved by the University of Maryland Institutional Animal Care and Use Committee (IACUC). PBMCs were obtained from healthy individuals and a total of 5 × 106 cells were inoculated by intraperitoneal route in each mouse to generate humanized mice. After 4 weeks of engraftment, blood was collected from retro-orbital sinus and stained for analysis using flow cytometry. Successful engraftment was determined by positive staining of mice whole blood with human CD45 antibody. Mice were then divided into groups of five and assigned to various treatment groups.
HIVBAL with a p24 concentration of 160 ng was used to infect mice via intraperitoneal route. This concentration was previously found to result in successful infection of all animals resulting in massive loss of CD4 T cells. Groups of uninfected and infected but untreated mice were controls. In treatment groups, mice were either treated with zoledronate-expanded cells, a combination of expanded cells and anti-HIV envelope antibody b12 or with b12 antibody alone.
Autologous PBMC were expanded by using a combination of zoledronic acid (zoledronate; Novartis, Basel, Switzerland) and recombinant human IL-2 (Tecin Biological Resources Branch, NIH, MD, USA) . During HIV infection, the numbers and function of Vγ9Vδ2 T cells are severely compromised and efforts to stimulate them with γδ stimulant, phosphoantigen isopentenyl pyrophosphate (IPP), are not successful. Aminobisphosphonate zoledronate overcomes this defect and leads to expansion of functionally active Vγ9Vδ2 T cells . Frequency of Vδ2 expressing cells was determined by flow cytometry and cultures with ˜90% Vδ2 expressing cells were used for adoptive transfers.
The role of ADCC in HIV is increasingly being recognized . Cells expanded with zoledronate have potent ADCC activity when used in combination with specific antibodies against tumors  and also against HIV envelope coated target cells in in vitro systems (data not shown). We attempted to use the expanded γδ T cells along with 10 mg/kg of b12 antibody to test this anti-HIV ADCC effect in vivo. The b12 monoclonal antibody is a broadly HIV neutralizing antibody, and has potent ADCC capacity .
Animals were infected 4 weeks after engraftment. Cell and antibody transfers were carried out at days 3, 7 and 10 after infection in relevant groups. A total of 15 × 106 zoledronate-expanded cells were injected at each time point per animal via lateral tail vein. At the same time, 250 μg of b12 antibody was injected in relevant groups by the same route. The dose of antibody and cell injections are based on published work in a mouse model using similar antibody/cell suspensions [19,20]. Blood was collected at various times after infection for flow cytometric evaluation of surface markers as well as for measurement of HIV core antigen p24 in plasma. Monoclonal antibodies were from BD biosciences unless otherwise specified. Whole blood was stained for cellular markers following lysis and fixing using a commercial kit (Beckman Coulter) and acquisition was done on an FACS Calibur. Data analysis was performed using FlowJo software (Tree Star, OR, USA). The p24 antigen was detected in plasma samples from individual mice at various times post-infection using a commercial ELISA kit.
Expanded cells were injected at days 3, 7 and 10 after infection with HIV. We are testing the potential of γδ T cells as an immunotherapy in individuals whose infection is detected in very acute stages. Accordingly, this regimen was adopted. Flow cytometry showed significant frequency of Vδ2+ cells (average 4.5% of T cells) in inoculated animals tested 7 days after adoptive transfer. This represents tenfold higher frequency compared with circulating Vδ2+ T cells (average Vδ2+ frequency 0.4% of T cells) in our humanized mouse model.
Severe depletion of HIV target cells expressing CD4 and CCR5 was observed in all infected groups by day 10 after infection (Figure 1) when compared with uninfected group. There were no improvements in frequency of this subset in animals inoculated with expanded cells and/or antibody compared with untreated groups by day 24, which was the last day of our experiments. Along with depletion of target cells, the CD4:CD8 ratio  was inverted in all infected groups irrespective of treatment (Figure 2). The levels of viral replication measured by detecting p24 antigen in plasma samples showed comparable p24 in all infected groups at most time points, with a peak replication observed in most animals at day 7. There was no decrease in viral load in any treated group compared with untreated infected group at any time tested (Figure 3).
While there was no benefit of γδ therapy in any infected animal in our system, we observed a significant spike in viral replication in animals that were injected with expanded cells as well as the anti-HIV envelope antibody b12 at week 2. We have previously tested the antibody-dependent cytotoxicity of cells expanded in this manner against HIV envelope coated target cells (data not shown) and were expecting a similar beneficial effect of such treatment against HIV in this animal model. One explanation for the unexpected result could be the Fc receptor (CD16) mediated immune activation caused by such a treatment and resulting spike in HIV replication. Cross-linking of CD16 on immune cells results in release of cytokines such as TNF-α as well as upregulation of activation markers like CD25 (data not shown). Similar changes in the microenvironment could possibly promote HIV replication. In an unexpected finding, recently it was shown that resting Vδ2+ cells act as reservoir for HIV  whereas HBMPP+IL-2 activated Vδ2+ cells could be readily infected with HIV JR-CSF in a CD4-dependent manner. Based on these findings, the study hypothesized that immune activation caused by HIV infection induces transient CD4 expression on this cell subset rendering them susceptible to HIV infection. Such a scenario is possible in our system where zoledronate +IL-2 expanded cells were injected during acute phase of infection.
Expanded Vδ2 cells have not been unambiguously shown to limit HIV in an in vivo model. Aminobisphosphonates such as zoledronate along with cytokines IL-2 or IL-15 are used to expand these cells, which then have potential for immunotherapy against cancers and some infectious diseases via direct cytotoxicity or antibody-mediated cytotoxicity. Recent success using such expanded cells to control influenza virus replication in a humanized mouse model encouraged us to test the possibility against HIV. We showed here that adoptive transfer of expanded γδ T cells either alone or in combination with anti-HIV envelope b12 antibody failed to suppress HIV replication in our system of acute HIV infection. It is possible that the concentration of HIV used for infection in our experiments is high and may have resulted in an uncontrollable infection. Previously, use of a lower dose of 120 ng/mouse resulted in a slower infection characterized by peak viral replication observed at week 2 instead of week 1 as well as significantly lower levels of p24 in plasma (data not shown). However, in that case similar to here, no effect of adoptive transfer of expanded cell on viral p24 levels or depletion of viral target cell was observed. It is possible that during acute HIV infection, the immune activation caused by such treatment counteracts the possible beneficial effect by providing susceptible cells for virus replication. These expanded Vγ9Vδ2 T cells are capable of producing pro-inflammatory cytokines that can potentially activate HIV replication. In a rhesus macaque SHIV model of HIV infection, injection of HMBPP+IL-2 during acute stage of infection resulted in an increase in virus replication whereas similar treatment during chronic stage had no effect on plasma viral loads . Administration of adoptive transfers of expanded cells with or without anti-HIV antibodies during set point viremia phase of infection thus may have different outcomes and will be tested in the future.
Adoptive transfer of zoledronate expanded γδ T cells do not suppress HIV replication in acute model of HIV infection.
Financial & competing interests disclosure
This work was funded in part by Institutional Grant of Institute of Human Virology of the University of Maryland (B Poonia) and by PHS grant AI110229 (B Poonia) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The author has obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.