article: Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis (Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.
  Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide among infectious diseases, and has become increasingly prevalent and deadly as a result of the emergence of multi-drug resistant (MDR) TB and HIV/AIDS pandemic [1], [2]. Given the possibility that TB patients usually have immune dysfunction, immune intervention regulating or enhancing anti-TB immune responses may be beneficial for clinical treatment of TB or MDR-TB. Although immune therapy or adjunctive immune plus antibiotics treatment has been long considered attractive for clinical treatment of TB, especially MDR TB, successful treatment modalities have not been identified due to the lack of an immune regimen that specifically targets immune components of anti-TB immunity. In fact, little is known about how human immune cells control a primary Mtb infection, despite that it is generally believed that human CD4+ T cells are important for resistance to TB [3], [4]. In-depth studies of Mtb-specific T effector cells may facilitate our understanding of anti-TB immune mechanisms and help identify potential immune intervention for drug-resistant TB. Vγ2Vδ2 T cells exist only in primates, constitute 65–90% of total human circulating γδ T cells, and regulate or contribute to both innate and adaptive-like immune responses in infections [5], [6], [7], [8], [9]. Vγ2Vδ2 T cells recognize phosphoantigen (E) -4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) produced by M. tuberculosis (Mtb) or other selected pathogens in TCR-dependent fashion [10], [11], [12], [13]. Our decades-long studies in non-human primate models contribute to illustrating biology and immune responses of human Vγ2Vδ2 T cells in Mtb and other infections [6]. Recently, we and others have developed a unique manipulating system to remarkably expand Vγ2Vδ2 T cells in vivo. We showed that administration of phosphoantigen compound plus IL-2 induced remarkable expansion of Vγ2Vδ2 T cells, antagonized cytokine-induced Foxp3+ Treg, reversed Treg-mediated immune suppression, and attenuated pneumonic plague lesions in non-human primates [14], [15], [16], [17]. These findings provide a rationale for conducting a proof-of-concept study to determine whether phosphoantigen/IL-2 administration can expand/differentiate Vγ2Vδ2 T cells, improve immune system and resist TB in non-human primates. Finding may shed light on γδ T-cell-targeted immune intervention potential using a clinical drug zoledronate capable of expanding Vγ2Vδ2 T cells, as this drug is being tested as γδ T-cell-based therapeutic regimen for treatment of metastatic cancers [18], [19]. Studies of Vγ2Vδ2 T cell-targeted intervention can only be done in primates, as there is no evidence that γδ T cells from mice and other animals can recognize phosphoantigen and other microbial antigens [6].
  To conduct the proof-of-concept study, macaques were treated intermittently with the Picostim (identical to HMBPP except one carbon difference) plus IL-2 regimen [15] at day −3 and day 15, respectively, after pulmonary Mtb infection. Picostim/IL-2 administration during Mtb infection of macaques reproducibly induced remarkable expansion of Vγ2Vδ2 T cells (Fig. 1A). To minimize variation in evaluating infection/immunity in out-bred macaques, we performed 3 serial repeated experiments and, in each in vivo experiment, the test group and 2 control groups were simultaneously investigated. Vγ2Vδ2 T cells were expanded up to 60% from base-line 1% of total CD3+ T cells or increased to absolute mean numbers from ∼40/ul to ∼2000/ul after Picostim/IL-2 administration (Fig. 1A). Notably, expanded Vγ2Vδ2 T cells were able to traffic to and accumulate in the pulmonary compartment during Picostim/IL-2 treatment and Mtb infection (Fig. 1B). Virtually, such pulmonary accumulation of phosphoantigen-activated Vγ2Vδ2 T cells was consistent with increases in these γδ T cells in lung interstitial tissues and pulmonary lymphoid follicles[[14], and data not shown]. In contrast, control IL-2 alone or saline/BSA treatment did not induce significant increases in numbers of Vγ2Vδ2 T cells in the circulation and pulmonary compartments (Fig. 1a, b). The repeated Picostim/IL-2 treatment at day 15 after Mtb infection led to a subtle increase in the mean number of blood Vγ2Vδ2 T cells compared to the level at day 9, the time in which numbers of Vγ2Vδ2 T cells remained higher than baseline despite declining from the peak expansion following the initial treatment (Fig. 1a, 1b). Thus, these results demonstrated that Picostim/IL-2 treatment during pulmonary Mtb infection of macaques induced major expansion of Vγ2Vδ2 T cells and pulmonary trafficking/accumulation of these cells. Since Picostim/IL2 regimen induced major expansion and pulmonary trafficking/accumulation of Vγ2Vδ2 T cells, we examined whether this immune intervention could lead to changes in bacterial burdens after pulmonary Mtb infection. We measured bacterial CFU counts in lung tissue homogenates from right caudal lung lobe (infection site), right middle lobe and left caudal lobe at the end point after Mtb infection. The Picostim/IL2-treated group exhibited lower numbers of bacilli organisms in lung tissue homogenates from all studied lung lobes than the saline/BSA-treated and IL2-treated control groups, particularly in right middle and left caudal lung lobes than IL-2-treated control group (Fig. 1c). These findings showed that Picostim/IL-2 administration in early Mtb infection could expand Vγ2Vδ2 T cells and lead to reduction in Mtb bacterial burdens in lungs. We then sought to determine whether Picostim/IL2 manipulation of Vγ2Vδ2 T cells would increase resistance to TB lesions in lungs after Mtb infection. We conducted complete necropsy and gross pathology studies at day 65 after Mtb infection to examine detectable resistance to TB pathology and lesions in the Picostim/IL2-treated group in comparisons with the control groups. Interestingly, Picostim/IL2-treated macaques exhibited an increased resistance to TB lesions in lungs compared to saline/BSA and IL2-treated controls. Virtually, four animals (IDs: 8022,8014,8013,8006) in Picostim/IL2-treated group did not show any gross TB lesions in lung tissues (Fig. 2, Fig. S1 in Text S1), three animals displayed localized TB lesions limited to the right caudal lobe (infection site) of lungs (Fig. 2), and only two macaques showed apparent TB lesions involving the right caudal and middle lung lobes or left lower lobe (Fig. 2, Fig. S1 in Text S1). In contrast, all control macaques treated with saline/BSA exhibited severe/extensive caseating and miliary TB lesions or caseation pneumonia, and displayed extensive coalescing granulomas in right caudal/middle lobes and left lower lobe (Fig. 2, Fig. S1 in Text S1). TB lesions in these saline/BSA-treated macaques were readily disseminated to the opposite lung, hilar lymph nodes, and pleural or extrathoracic organs. All macaques except one in the IL2-treated control group developed TB lesions, and the lesions were more apparent than those in the Picostim/IL2-treated group (Fig. 2, Fig. S1 in Text S1). When the gross TB pathology or lesions was evaluated in details and compared between groups using the scoring system as we and others previously described [20], [21], [22], we found that the Picostim/IL2-treated group exhibited significantly milder TB lesions or pathology than saline/BSA-treated and IL2-treated control groups (Fig. 3a). Of note, the immune resistance to TB lesions after Picostim/IL2 treatment did not appear to be mediated by IL2 since Picostim/IL2-treated macaques showed less severe TB lesions or pathology than the IL2 alone group did (Fig. 3a). IL2 alone conferred detectable resistance to TB lesions compared to BSA/saline, but could not effectively reduce Mtb bacterial burdens ([22], and Fig. 1c, Fig. 3a), whereas Picostim/IL2 expansion of Vγ2Vδ2 T effector cells decreased levels of Mtb infection (Fig. 1c). Consistently, histopathology analyses also implicated immune resistance to TB in Picostim/IL2-treated macaques. TB granulomas in all saline/BSA-treated control macaques were apparently extensive, coalescing and widely necrotic in tissue sections from all studied lung lobes (Fig. 3b, Fig. S2 in Text S1). TB granulomas were seen in lung tissue sections from all IL2-treated macaques although these animals were less necrotic than saline/BSA-treated group (Fig. 3b, Fig. S2 in Text S1). Interestingly, among those four Picostim/IL2-treated macaques that did not show any gross lung TB lesions at necropsy, three macaques (IDs: 8022,8014,8013) did not exhibit any detectable microscopic TB granulomas in the lung tissue sections derived from right caudal and other lung lobes despite that we employed digital microscopic scanning for extensive analyses (Fig. 3b, Fig. S2 in Text S1). One of (ID: 8006) those four animals showed only a few tiny, lymphocytic, non-necrotic microscopic granulomas (Fig. 3b). In other five Picostim/IL2-treated macaques, microscopic TB granulomas were smaller, more focal and much less necrotic than those in the saline/BSA-treated macaques, and similarly non-necrotic or more lymphocytic compared to IL2-treated macaques (Fig. 3b, Fig. S2 in Text S1). Taken together, the gross pathology and histopathology results suggested that Picostim/IL2 expansion of Vγ2Vδ2 T cells could confer immune resistance to TB after pulmonary Mtb infection. We then undertook mechanistic studies to examine if the immune resistance to TB coincided with anti-TB effector function of Vγ2Vδ2 T cells after Picostim/IL2 administration. Since mouse IFNγ and perforin are needed for anti-TB immunity [23], [24], [25], [26] and human granulysin kills Mtb [4], we examined if these anti-TB cytokines could be produced by Picostim-expanded Vγ2Vδ2 T cells in the pulmonary compartment. We employed both the conventional intracellular cytokine staining (ICS) upon in vitro antigen stimulation and the modified direct ICS assay without in vitro antigen stimulation since the direct ICS assay had been demonstrated to be useful for measuring T effector cells that were close to the in vivo setting during infections [22], [27], [28], [29], [30]. We found that appreciable numbers of Vγ2Vδ2 T cells in the pulmonary compartment were able to re-recognize HMBPP phosphoantigen and produce IFNγ over time after the Picostim/IL-2 administration (Fig. 4a). In fact, Picostim/IL2-expanded Vγ2Vδ2 T cells could produce IFNγ constitutively without the need for in vitro HMBPP phosphoantigen stimulation (Fig. S3a in Text S1). Similarly, pulmonary Vγ2Vδ2 T effector cells were able to produce perforin in the presence or absence of HMBPP stimulation, and these effectors peaked at ∼1 week in BAL cells after Mtb infection (Fig. 4a, Fig. S3a in Text S1). Furthermore, in-situ confocal imaging studies showed that Vγ2Vδ2 T effector cells producing perforin or granulysin were detectable in lung tissue sections derived from the Picostim/IL2-treated macaques (Fig. 4b, Fig. S3b in Text S1). Virtually, some of activated Vγ2Vδ2 T cells in lung tissues from Picostim/IL2-treated macaques were able to co-produce perforin and granulysin in situ (Fig. 4b, Fig. S3b in Text S1). In contrast, saline/BSA- or IL-2-treted control macaques did not show any perforin/granulysin-co-producing Vγ2Vδ2 T cells in lung tissues although perforin-producing cells (non-Vγ2) were detectable in IL-2-treated macaques (Fig. 4b). Immunohistochemistry analysis also demonstrated that Picostim/IL2-treated macaques exhibited more Vγ2 T cells in lung parenchyma and granulomas tissues than control IL2-treated and saline/BSA-treated animals even at the end time point (Fig. S3c in Text S1). Given that Picostim-differentiated Vγ2Vδ2 T cells produced anti-Mtb cytokines, we presumed that expanded Vγ2Vδ2 T effector cells could limit Mtb replication during Mtb infection. To test this hypothesis, we used the effector T cell-cloning technique as we previously described [31] to generate phosphoantigen-activated Vγ2Vδ2 T effector cells from PBL of Mtb-infected macaques [21] and assessed them for the ability to regulate intracellular Mtb replication. These Vγ2Vδ2 T effector cells expressed granulysin and perforin, and could limit Mtb growth in Mtb-infected monocytes (Fig. 5a). Consistently, some Vγ2Vδ2 T cells that expanded and differentiated in vivo at 14 days after Picostim/IL-2 treatment could recognize Mtb-infected autologous macrophages leading to inhibition of intracellular Mtb growth, and such inhibition could be reduced by antibodies against granulysin/perforin (FigS. 3d). The result indicated the need of granulysin and perforin secretion for anti-Mtb effector function and confirmed what was described in the in vitro study of human γδ T effector cells [32]. To examine a molecular mechanism whereby Picostim/IL2-expanded Vγ2Vδ2 T effector cells inhibited Mtb replication, we sought to determine whether macaque granulysin or perforin produced by Picostim-expanded Vγ2Vδ2 T effector cells exerted anti-Mtb activities. To this end, granulysin (Gnl) and active domain of perforin (PC) were expressed and purified using the E. coli expression system (Fig. S4 in Text S1, [33]), and then evaluated for the ability to affect extracellular and intracellular Mtb replication. We designed and produced active component of perforin [34], because the whole protein of macaque perforin was not optimally expressed and secreted in the E. coli expression system (data not shown). The active macaque granulysin killed extracellular Mtb at a concentration of 50 uM (Fig. 5b, left), and inhibited intracellular Mtb in monocytes/macrophages in the presence of active PC (Fig. 5b, right). The Fig. 5b data derived from the new system producing macaque granulysin/perforin help to explain anti-Mtb effector function of Vγ2Vδ2 T cells while confirming the result from the earlier study of human granulysin [35]. Taken together, these results consisted with the resistance to TB in Picostim/IL2-treated macaques, supporting the view that Vγ2Vδ2 T effector cells could limit intracellular Mtb replication during Mtb infection via production of granulysin and perforin. Recent in vitro studies have shown that activated human Vγ2Vδ2 T cells can function as APC presenting peptide antigens to αβ T cells [36]. However, whether Vγ2Vδ2 T effector cells can enhance anti-microbial responses in vivo remains poorly characterized. As an additional mechanistic study, we investigated whether in vivo expansion of Vγ2Vδ2 T cells after Picostim/IL-2 treatment could augment immune responses of peptide Ag-specific αβ T cells in the context of immune resistance to Mtb infection. To this end, we compared frequencies of mycobacterium-specific CD4+ and CD8+ T effector cells producing anti-TB cytokine IFNγ between Picostim/IL2-treated and control groups of macaques since phosphoantigen was highly specific for Vγ2Vδ2 T cells, but not for CD4+ or CD8+ T cells. Picostim/IL2 expansion of Vγ2Vδ2 T cells did not lead to significant increases in frequencies of PPD-specific IFNγ-producing αβ T effector cells in the blood when compared to those from IL-2-treated group (data not shown). However, Picostim/IL2-treated macaques exhibited significantly greater numbers of Mtb-specific IFNγ-producing CD4+ T effector cells in the pulmonary compartment than control macaques treated with IL2 alone or saline/BSA during early phase of Mtb infection (Fig. 6a). Picostim/IL2 immune intervention also promoted greater increases in pulmonary PPD-specific IFNγ-producing CD8+ T effector cells than IL2 alone and saline control treatment (Fig. 6a). Moreover, an increased ability of CD4+/CD8+ T cells to constitutively produce IFNγ in the setting relevant to the in vivo condition was also seen in Picostim/IL2-treated macaques, as we detected an increased number of pulmonary CD4+ and CD8+ T effector cells capable of producing IFNγ without the need for peptide Ag stimulation in vitro (Fig. S5 in Text S1). We certainly realized that the pulmonary mobilization of these effector cells could not directly prove one of the immune mechanisms whereby expanded γδ T cells contribute to anti-TB immunity. Since CD4+/CD8+ Th1 immune responses were often initiated and promoted by IL-12 production, we examined if Picostim-expanded Vγ2Vδ2 T effector cells were able to produce IL-12 leading to the enhanced pulmonary Th1 responses in Picostim/IL-2-treated macaques. Interestingly, we detected significant numbers of Vγ2Vδ2 T effector cells capable of constitutively producing IL-12 without the need for in vitro HMBPP stimulation after Picostim/IL2 administration, but not saline/BSA or IL2 alone treatment (Fig. 6b). These results suggested that Picostim/IL2-expanded Vγ2Vδ2 T cells were able to constitutively produce IL-12, and their expansion/differentiation led to enhanced pulmonary immune responses of Mtb peptide-specific αβ CD4+/CD8+ T effector cells.
  Despite that human γδ T cells have been discovered for more than 25 years, the role of Mtb-specific Vγ2Vδ2 T cells in immunity against TB and other infectious diseases has not been defined in humans. Data from the current proof-of-concept study suggest that Picostim/IL-2 immune intervention during pulmonary Mtb infection can expand/differentiate Vγ2Vδ2 T effector cells, limit Mtb replication/dissemination, and attenuate TB lesions in non-human primates. Earlier negative finding for γδ T cells in mouse TB model does not appear to be conclusive [26], as there is no evidence that mouse γδ T cells can recognize Mtb phosphoantigen or other antigens [6]. Now we present the first in vivo evidence suggesting that phosphoantigen/IL2 expansion of Vγ2Vδ2 T cells can induce immune resistance to severe TB. This finding extends in vitro data demonstrating anti-Mtb effector function of human γδ T cells [8], [37], [38], [39], and appears to be novel and relevant to elucidating in vivo function of human Vγ2Vδ2 T cells. Picostim/IL2 expansion/differentiation of Vγ2Vδ2 T effector cells mimics the natural immune stimulation driven by Mtb phosphoantigen and IL2 in Mtb infection. The Picostim/IL2 intervention simply moves the selective expansion/differentiation of Vγ2Vδ2 T effector cells earlier and to a greater extent, since production of phospohoantigen and IL2 will become prominent only in a later phase of Mtb infection. In fact, Mtb infection or BCG vaccination can induce remarkable expansion of Vγ2Vδ2 T cells at ∼3–4 weeks postinfection, and rapid recall expansion of Vγ2Vδ2 T cells in BCG-vaccinated macaques correlates with the vaccine-induced protection against life-threatening TB [9]. Earlier and greater-magnitude expansion of Vγ2Vδ2 T cells during Mtb infection appears to create a proof-of-concept setting in which to examine immune function of Mtb-specific Vγ2Vδ2 T effector cells in primates, as rapid clonal expansion and pulmonary trafficking of Mtb-specific CD4+/CD8+ T cells have been interpreted as important immune events in immunity against TB in macaques [40]. The resistance to TB after phosphoantigen treatment could be due to killing and blunting of Mtb as they enter the pulmonary compartment after infection, since the treatment is given during early Mtb infection rather than established TB disease. It should be pointed out that only some of expanded Vγ2Vδ2 T cell subpopulations are protective although almost all γδ T cells expressing Vγ2Vδ2 TCR have been shown to recognize Mtb phosphoantigen [6], [10], [39]. Consistent with this concept, we and others including Spencer/Hoft et al have repeatedly shown that only certain percentages of expanded Vγ2Vδ2 T cells can differentiate into effector cells capable of producing anti-TB cytokines or inhibiting mycobacterial growth during phosphoantigen/IL2 treatment or Mtb infection. It is also noteworthy that phosphoantigen/IL2 treatment of chronic TB may not be as efficacious as the treatment of early-phase or primary TB, since patients with chronic TB often exhibits anergy or insufficiency of γδ T cell effector function [6]. In addition, an in vitro study by Spencer/Hoft et al showed that large numbers of phosphoantigen-activated γδ T cells could not recognize BCG-infected target cells [39]. The immune resistance to Mtb infection and TB appears to result from multi-effector functions of phosphoantigen-expanded Vγ2Vδ2 T cells rather than IL-2 immune activation since Picostim/IL2-treated macaques exhibited lower Mtb CFU counts and milder TB lesions in most lung lobes than IL2-treated controls. It is noteworthy that IL-2 treatment confers resistance to TB lesions compared to BSA/saline treatment [22]. Notably, IL-2 administration predominantly expanded CD4+CD25+Foxp3+ Treg [15], whereas Picostim/IL-2 regimen massively expanded Vγ2Vδ2 T cells in mycobacterial infection, down-regulated IL2-induced expansion of IL2-induced Treg cells and reversed Treg-associated immune suppression in vivo [15]. Although IL2 alone is protective against TB lesions, the regimen could not effectively reduce Mtb bacterial burdens ([22], Fig. 1c, Fig. 3a). Picostim/IL2 expansion of Vγ2Vδ2 T cells led to a decreased level of Mtb infection and more apparent resistance to TB than IL-2 expansion of Treg and T effectors ([22], Fig. 1c, Fig. 3a). Of note, all IL2-treated macaques except one developed TB lesions. In contrast, approximately 45% of phosphoantigen/IL2-treated macaques did not exhibit detectable TB, and gross pathology scores in phosphoantigen/IL2-treated group are significantly smaller than those in IL2-treated and saline/BSA-treated control groups. While anti-Mtb effector function of phosphoantigen-expanded Vγ2Vδ2 T cells appears to be relevant to controlling Mtb infection and attenuating TB, the ability of Vγ2Vδ2 T effectors to mediate homeostasis against inflammation and tissue damage may also contribute to the immune resistance to TB. This notion is supported by our recent observation that phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells can produce a homeostatic mediator FGF-7, and coincide with attenuation against pneumonic plague damages of lung tissues after pulmonary infection even with extracellular Y. pestis [14], whose protection often requires neutralizing Ab. Data from the mechanistic experiments suggest that phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells producing granulysin/perforin during Mtb infection may contribute to the in vivo function restricting Mtb infection or dissemination while these expanded γδ T cells are actively mounting anti-TB Th1-like IFNγ cellular responses. The phosphoantigen/IL2-expanded Vγ2Vδ2 T cells appear to differentiate into multi-functional effector cell subpopulations that are able to mount anti-Mtb effector functions via producing at least IFNγ, perforin and granulysin. It is noteworthy that Vγ2Vδ2 T effector cells producing granulysin/perforin are able to limit Mtb growth in macrophages. The capability of Vγ2Vδ2 T effector cells to co-produce granulysin and perforin seems to be necessary for anti-Mtb effector function of Vγ2Vδ2 T effector cells as macaque granulysin, like human counterpart, exerts bactericidal effect on extracellular Mtb and inhibits intracellular Mtb in presence of perforin [35]. Earlier human studies showed that potent in vitro stimulation of human Vγ2Vδ2 T cells allowed these highly-activated γδ T cells to inhibit Mtb growth and that anti-perforin/anti-granulysin antibodies could reduce the anti-Mtb effector function [8], [32], [37], [39]. Now, the current study extended the previous in vitro work by demonstrating both the in vivo co-production of granulysin/perforin by Vγ2Vδ2 T effector cells in lung tissues and the direct anti-Mtb activities exerted by these two cytokine proteins. Our findings support the notion that anti-Mtb Vγ2Vδ2 T effector cells and granulysin/perforin effector cytokines might contribute to phosphoantigen/IL2-induced resistance to Mtb infection or TB lesions, although down-regulation of Treg for enhancing immune responses might also play a role. Thus, phosphoantigen-differentiated Vγ2Vδ2 T effector cells and in vivo co-production of granulysin/perforin in lungs may be relevant to a mechanism underlying the in vivo control of Mtb replication or dissemination in Picostim/IL2-treated macaques. Another surprising observation is that Picostim/IL2-expanded Vγ2Vδ2 T cells are able to constitutively produce IL-12 without Ag stimulation, and expansion of Vγ2Vδ2 T cells leads to enhanced pulmonary immune responses of Mtb-specific αβ T effector cells. IL-12, a major cytokine critical for eliciting Th1 immune responses, is usually produced by DC or other APC, but not T cells. Our demonstration of IL-12 production by Vγ2Vδ2 T effector cells is therefore considered novel. The constitutive production of IL-12 by Vγ2Vδ2 T effector cells helps to explain indirectly how expansion/differentiation of Vγ2Vδ2 T effector cells by phosphoantigen/IL2 could promote pulmonary Th1-like immune responses of CD4+ and CD8+ T cells in Mtb infection. In fact, frequencies of pulmonary CD4+ and CD8+ Th1-like cells in Picostim/IL2 group are significantly higher than those in saline/BSA or IL2 alone control during primary Mtb infection. The reason why enhanced responses were seen prominent only in pulmonary compartment but not in blood may be due to the fact that pulmonary Mtb infection leads to dominance of immune responses in lung but not in peripheral blood [40]. These results suggest that phosphoantigen expansion of Vγ2Vδ2 T effector cells may serve as immune adjuvant enhancing CD4+ and CD8+ Th1 responses. The finding also suggests that early expansion/differentiation of Vγ2Vδ2 T cells by phosphoantigen/IL2 during Mtb infection may not only limit Mtb replication and dissemination, but also help promote primary anti-TB CD4+ and CD8+ Th1 responses. The surprising in vivo observation of Vγ2Vδ2 T cell-driven anti-TB immunity would open an avenue for further mechanistic studies. To our knowledge, the current study also represents the first appreciable attempt that amplification and differentiation of a potentially protective Ag-specific T-cell subpopulation in Mtb infection can confer detectable efficacy of resistance to TB in primates. Since non-human primates resemble their human counterparts in biology and host responses to TB, our findings may help to rationalize future studies for evaluating γδ T cell-targeted treatment modality in chronic TB. Notably, human Vγ2Vδ2 T cells can be selectively expanded in vivo by zoledronate, a clinical drug widely used for treatment of bone-related disease, and that zoledronate/IL-2 expansion of Vγ2Vδ2 T cells has been tested as therapeutic regimen for treatment of metastatic cancers [18], [19]. Thus, specific phosphoantigen/IL2 immune stimulation early during Mtb infection rapidly expands and differentiates Vγ2Vδ2 T cells into multi-functional effector cells capable of mounting anti-TB IFNγ response, inhibiting Mtb growth through in vivo or constitutive production of perforin/granulysin, and producing IL-12 and promoting pulmonary Th1 responses of peptide-specific CD4+ and CD8+ T cells. The observation that phosphoantigen/IL2 expansion/differentiation of Vγ2Vδ2 T cells increases resistance to TB in nonhuman primates provides a rationale to design further studies of treatment of established TB. Such future studies might help guide single or adjunctive phosphoantigen expansion/differentition of Vγ2Vδ2 T cells for treatment of MDR-TB or AIDS-related TB in humans.
  The use of macaques and experimental procedures were approved by Institutional Animal Care and Use Committee and Biosafety Committee (Protocols A 05-072 and A 08-029), University of Illinois College of Medicine at Chicago (UIC), and we followed the national and international guidelines [International Primatological Society (IPS) International Guidelines for the acquisition, care and breeding of nonhuman primates] regarding “The use of non-human primates in research” to minimize potential suffering of the studied macaques. Daily or weekly clinical follow-up were taken to ensure that animals were not suffering from severe coughing, respiratory distress, depression, refusing to take food, body-weight loss or other potential life-threatening signs. Humane euthanization procedures were immediately taken if those signs occurred progressively. Twenty-seven cynomolgus macaques (M. fascicularis), 4–8 year old and 3–5 kg, were used in this study. All animals were maintained and used in accordance with guidelines of the institutional animal care and use committee (IACUC). Animals were anesthetized with 10 mg/kg ketamine HCl (Fort Dodge Animal Health, Fort Dodge, IA) i. m. for infection, bronchoalveolar lavage (BAL), blood samplings and treatments. All experimental protocols were approved by IACUC and IBC in University of Illinois Chicago. Picostim and HMBPP phosphoantigen chemicals are very similar in bioactivity and structure as they differ in only one atom linking the carbon chain and the phosphate moiety [15]. Both Picostim and HMBPP are specifically recognized by TCR expressed on primate Vγ2Vδ2 T cells [13] not by other γδ T cell subsets or αβ T cells. Picostim (>98 pure) was synthesized and provided by the company Innate Pharma (France). In the Picostim/IL2 co-treated group (n = 9), Picostim (25 mg/kg) was administered i. m. , as we previously reported [15], on day −3 and day 15, respectively, after Mtb infection; recombinant human IL-2 (rhIL-2; Proleukin, Chiron, Emeryville, CA) was given i. m. at a dose of 1. 2 million IU IL-2 once daily for 5 consecutive days beginning on the day of Picostim treatment. As a control group (n = 9), 1. 2 million IU IL-2/day was given once daily i. m. for 5 consecutive days. As another control (n = 9), five animals received saline and four received bovine serum albumin (BSA, 27. 2 ug) in 0. 5-ml s. c. for 5 consecutive days. All treated macaques did not exhibit any notable side effects during treatments.
summary: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of morbidity and mortality worldwide. While CD4+/CD8+ T cells are protective, role of γδ T cells in TB and other infections remains unknown in humans. Vγ2Vδ2 T cells exist only in primates, represent a dominant circulating γδ T-cell subpopulation, and recognize phosphoantigen from Mtb and some selected pathogens. Here, we determined whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection increased resistance to TB in macaques. Phosphoantigen plus IL-2 administration induced expansion and pulmonary accumulation of Vγ2Vδ2 T cells, significantly reduced Mtb counts and attenuated TB lesions in lung tissues. Expanded Vγ2Vδ2 T cells produced anti-TB cytokines IFNγ, perforin and granulysin, and co-produced perforin and granulysin in lung tissue. Perforin/granulysin-co-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin killed Mtb bacteria, and inhibited intracellular Mtb in presence of perforin. Furthermore, expansion of Vγ2Vδ2 T effectors enhanced pulmonary responses of peptide-specific CD4+/CD8+ T cells, which correlated with the ability of Vγ2Vδ2 T effector cells to produce IL-12. These results provide first evidence implicating a protective role of Vγ2Vδ2 T effector cells in TB, supporting a rationale to explore Vγ2Vδ2 T-cell-targeted treatment of drug-resistant TB or HIV-related TB.
keywords: immunity to infections
  immunology
  immunity
  biology
title: Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates