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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2018 Mar 2;24(6):1152–1162. doi: 10.1016/j.bbmt.2018.02.023

Early reconstitution of NK and γδ T cells and its implication for the design of post-transplant immunotherapy

Moniek A de Witte 1,2, Dhifaf Sarhan 1, Zachary Davis 1, Martin Felices 1, Daniel A Vallera 3, Peter Hinderlie, Julie Curtsinger 4, Sarah Cooley 1, John Wagner 5, Jurgen Kuball 2,6, Jeffrey S Miller 1
PMCID: PMC5993609  NIHMSID: NIHMS957872  PMID: 29505821

Abstract

Relapse is the most frequent cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HCT). NK cells and γδ T cells reconstitute early after allo-HCT, contribute to tumor immunosurveillance via MHC independent mechanisms and do not induce graft-versus-host disease (GVHD). Here we performed a quantitative and qualitative analysis of the NK and γδ T cell repertoire in healthy individuals, recipients of HLA-matched sibling or unrelated donor allo-HCT (MSD/MUD-HCT) and umbilical cord blood-HCT (UCB-HCT). NK cells are present at high frequencies in all allo-HCT recipients. Immune reconstitution (IR) of vδ2+ cells depended on stem cell source. In MSD/MUD-HCT recipients, vδ2+ comprise up to 8% of the total lymphocyte pool, whereas vδ2+ T cells are barely detectable in UCB-HCT recipients. Vδ1+ IR was driven by CMV reactivation and was comparable between MSD/MUD and UCB-HCT. Strategies to augment NK cell mediated tumor responses, like IL-15 and antibodies, also induced vδ2+ T cell responses against a variety of different tumor targets. Vδ1+ γδ T cells were induced less by these same stimuli. We also identified elevated expression of the checkpoint inhibitory molecule TIGIT (T cell Ig and ITIM domain), which is also observed on tumor-infiltrating lymphocytes and epidermal γδ T cells. Collectively, these data show multiple strategies which can result in a synergized NK and γδ T cell anti-tumor response. In the light of recent developments of low-toxicity allo-HCT platforms, these interventions may contribute to the prevention of early relapse.

Keywords: NK cells, gamma/delta T cells, immune reconstitution, hematopoietic transplantation

Introduction

Treatment-related mortality (TRM) has declined with the use of less intense conditioning regimens over the past several decades; however, relapse remains the most frequent cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HCT)1. After allo-HCT, patients are exposed to a period of immunosuppression to facilitate engraftment and minimize the risk of GVHD. Especially after reduced intensity conditioning, where a direct anti-tumor effect of the conditioning chemotherapy is less likely, achieving sufficient tumor control early after allo-HCT remains a challenge1.

Immune reconstitution (IR) is one of the critical parameters necessary for the successful use of allo-HCT2. Both NK cells as well as γδ T cells are recognized for their roles in immunosurveillance against cancer3, 4. Natural Killer (NK) cells start reconstituting within the first weeks after cell transfer and are amongst the first allogeneic immune cells present5. Similarly, γδ T cells, a subpopulation of CD3+ T cells characterized by a γδ TCR, reconstitute early after allo-HCT6. This is in contrast to αβ T cells which are slow to recover, leading to immunoincompetence in the first post-transplant months2. NK cells and γδ T cells share a range of features which make them attractive targets for enhancing the Graft-versus-Leukemia (GVL) effect of an allo-HCT7. In addition to early IR, both lymphocyte subsets recognize tumor cells in an MHC-independent fashion without an association with graft-versus-host disease (GVHD)7. Strategies aiming to broaden the innate repertoire while retaining functionality of these innate subsets are required to generate a more potent immune system during the first months post allo-HCT8.

CMV reactivation is a common complication after allo-HCT9. During primary CMV infection, NKG2C+ NK cells expand and become strong producers of IFNγ. Subsequently, increased frequencies of ‘adaptive NK cells’ (defined as CD56dimCD57+NKG2C+) persist, which can expand and become functionally active upon CMV reactivation10. We have recently shown that allo-HCT recipients with reactivated CMV had lower leukemia relapse associated with increased adaptive NK cells11. This outcome is in line with other reports showing a protective effect of CMV reactivation on relapse12, 13. Vδ1+ T cells are the primary γδT cell subset proliferating upon CMV infection/reactivation6, 14, 15. CMV-induced vδ1+ T cells are capable of recognizing both CMV-infected cells as well as primary leukemic blasts16, 17. Increased numbers of γδ T cells have also been associated with decreased relapse rates18.

Bisphosphonates (including zoledronate [ZOL]) can stimulate vγ9δ2+ T cells. These vδ2+ T cells are the largest circulating γδ T cell subset. Vγ9δ2+ T cells target tumor cells by sensing elevated levels of isopentenyl pyrophosphate (IPP) from the dysregulated mevalonate pathway (MVA)19. Bisphosphonates disrupt the MVA pathway, which results in an accumulation of IPPs in many transformed and infected cells. This subsequently leads to a conformational change of the ubiquitously expressed surface protein butyrophilin A1 (BTN3A1), the ligand for the Vγ9δ2 TCR19-21. The effects of bisphosphonates on NK cells are less well characterized and are thus far restricted to indirect stimulation of NK cells8.

IL-15 is a potent cytokine for NK cell development, expansion and homeostasis22. An attractive quality of IL-15 is that it does not induce proliferation of regulatory T cells (Tregs) 23. In γδT cells, IL-15 is important for development, maturation and function24-27. Not surprisingly, NK cells and γδT cells were the most responsive lymphocyte subsets in the first clinical trial using recombinant IL-15 (rh-IL-15) in cancer patients28.

NK cells highly express CD16 (FcγRIII), which allows NK cells to perform antibody-dependent cytotoxicity (ADCC). Additionally, γδT cells express CD16, albeit at much lower and more heterogeneous levels as compared to NK cells29. NK cells are main contributors to the anti-tumor effect seen with the first FDA-approved monoclonal antibody rituximab30. To further enhance NK cell mediated ADCC, bi-and trispecific killer engagers (BiKEs and TriKEs) have been developed. They bind CD16 at higher affinity than a natural Fc binder, creating a specific immune synapse between NK cells and tumor cells31. We have designed the 161533 TriKE, which can target CD33+ malignancies. The IL-15 linker promotes expansion, activation and enhances survival of NK cells32. In comparison to the 1633 BiKE, the 161533 TriKE showed better NK cell responses both in patient-derived NK cells as well as in murine models32. Also, γδ T cells are shown to perform ADCC with trastuzumab and with antibodies targeting B cell malignancies33-36.

IR depends on multiple factors, including stem cell source, graft manipulation, conditioning and post-transplant immunosuppression. Combined analysis of reconstituting NK and γδ T cells is not routinely performed37. Here we compare IR of NK and γδ T cells after allo-HCT in recipients of an HLA-matched sibling or unrelated donor (MSD/MUD-HCT) or umbilical cord blood (UCB-HCT) allografts. We show that the NK and γδ T cell composition early after MSD/MUD-HCT is comparable to healthy donors (HD) but different from UCB-HCT, with higher numbers of engrafting NK cells but significantly fewer γδ T cells. We identify multiple stimuli capable of activating reconstituting NK and γδ T cells that may lead to strategies to induce innate mediated tumor immune responses to prevent early relapse after allo-HCT.

Material and Methods

Healthy donors

Peripheral blood mononuclear cells (PBMCs) from healthy CMV-seronegative and seropositive donors were obtained from Memorial Blood Bank (Minneapolis, MN). All samples were de-identified before receipt, and the University of Minnesota institutional review board approved used in accordance with the Declaration of Helsinki.

Patient data

The University of Minnesota Blood and Marrow Transplant program prospectively collected all data regarding patient characteristics, immune monitoring and outcomes. The University of Minnesota institutional review board approved all protocols, and all patients (and/or their legal guardians) provided informed consent in accordance with the Declaration of Helsinki. Samples from patients with various malignant hematological diseases were analyzed for immune reconstitution (IR). Patients with relapse < 3 months and aGVHD grade IV were excluded for analysis. GVHD prophylaxis was still present in most subjects at the 2-3 month sample time in the UCB group (n=9 cyclosporine (CSA)/mycophenolate mofetil [MMF] and n=2 CSA/sirolimus) and the MSD/MUD group (n=8 CSA/MMF, n=1 CSA/MMF/Tacrolimus, n=3 CSA/methotrexate[MTX], and n=2 MTX/Tacrolimus). Prior and weekly after transplantation, recipients were assessed for CMV exposure. Only CMV seronegative patients were selected for the cohort which did not show a CMV reactivation. Patient characteristics are shown in Supplementary Table 1.

Flow cytometry analysis

Frozen PBMCs were thawed and rested overnight. When indicated, recombinant IL-15 (10 ng/ml) was added to the cells. For phenotypical analysis, cells were stained with fluorochrome-conjugated antibodies detailed in Supplementary Table 2. The gating strategy to dissect NK cells, γδ T cell subsets and αβ T cell subsets is depicted in Supplementary Figure 1. All cells were acquired by LSRII and analyzed by FlowJo 10.0.

Functional analysis

For functional analysis, frozen PBMCs were thawed and rested overnight. When indicated, recombinant IL-15 (10 ng/ml) was added to the cells. The tumor cell lines K562, Raji, HL60 and THP-1 were used as target cells. When indicated, tumor cells were incubated overnight with Zoledronate (Enzo Life Sciences) at a concentration of 20 μM. Degranulation and cytokine production were evaluated following 6 hours of incubation at an E:T ratio of 2:1. Rituximab (10 μg/ml) or 161533 TriKE32 (30 nM) were added at the start of the function assay. To detect degranulation, anti-CD107a was added at the start of the function assay. Cells were treated with the protein transport inhibitors Golgistop and Golgiplug (BD Biosciences) 1 hour after the start of the functional analysis. Analysis of TNFα and IFN-γ production was performed following fixation and permeabilization with 0.1% Triton X-100.

Statistical analysis

All data were first analyzed using the software FlowJo 10.0 and summarized by Prism Version 6 software (GraphPad). Differences and correlations among groups were analyzed using the Mann-Whitney test or Spearman Correlation test as indicated in the figure legends.

Results

NK and γδ T cell repertoire early post allo-HCT is impacted by stem cell source and CMV reactivation

In this study, we quantitatively and qualitatively characterized the NK and CMV repertoire in 2 cohorts of allo-HCT recipients (Supplementary Table 1) and compared those to healthy donors (HD). The MSD/MUD cohort represents recipients of an HLA-matched related or unrelated HCT. The other cohort represents recipients of UCB HCT. To assess whether early IR of the NK and γδ T cell repertoire was impacted by stem cell source, frozen peripheral blood (PB) samples were analyzed at 2-3 months after allo-HCT (median 68 days post HCT, range [54 – 106] after UCB-HCT [n=26] or MSD/MUD-HCT [n=28]) (Figure 1). At the time of the 2-3 month sample, subjects where still on similar GVHD prophylaxis and donor engrafted in the myeloid and lymphoid lineage. NK cell percentages were higher after transplant as compared to samples from HD, with the most expansion in UCB recipients, where NK cells comprised 29% (75 – 4.6) of the lymphocyte population. Compared to NK cells, γδ T cells were present in significantly lower percentages. In recipients of MSD/MUD-HCT, the γδ T cell repertoire was comparable to HD (Figure 1A). By contrast, after UCB-HCT, γδ T cells were detectable only in a small number of patients. We subsequently separated γδT cells in vδ1+ T cells, vδ2+ T cells and vδ1-vδ2- γδT cells. Normally, Vδ2+ T cells are the largest circulating γδ T cell fraction38. Upon CMV exposure, frequencies of vδ2- T cells in PB can significantly increase38, 39. Here we found that Vδ2+ T cells are the predominant γδ T cell fraction in HD as well as after MSD/MUD-HCT, whereas in UCB recipients, vδ2 cells are virtually absent (Figure 1A). Vδ1+ T cells are present at low frequencies in most patients after allo-HCT, but in contrast to vδ2+ T cells, v δ1+ T cells and vδ1-vδ2- T cells could clearly be detected both after MSD/MUD-HCT as well as after UCB-HCT.

Figure 1. NK and γδ T cell repertoire in healthy donors (HD) and patients after matched sibling donor (MSD)/ matched unrelated donor (MUD) or umbilical cord blood (UCB) transplant.

Figure 1

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A) Analysis of NK cells, γδ T cells and γδ T cell repertoires in blood of healthy donors (n=47; triangle), in blood of patients 2-3 months after MSD/MUD transplant (n=28; square) or UCB transplant (n=26; circle). Values represent the percentage of the indicated populations gated on total lymphocytes. B) The percent of γδ T cells is shown for individual subjects of each cohort. C: The correlation between the percentage γδ T cells (left) and vδ2 T cells (right) in donors and recipient of MSD/MUD transplantation. Statistics: Mann-Whitney test: (* P<.05; ** p<0.01; **** p<0.0001); Correlation: Spearman, 2-sided.

When γδ T cell percentages are visualized as individual ascending values, 30% of HD and MSD/MUD-HCT recipients have relatively high γδ T cell populations, with percentages up to 10% of the total lymphocyte population. However, in two-thirds of individuals, γδ T cells account for a minor fraction of circulating lymphocytes. After UCB-HCT, few patients reconstituted with γδ T cells (Figure 1B). In 12 MSD/MUD recipients, we could pair γδ T cell repertoires with donor samples. For γδ T cells and vδ2 T cells, we found a clear correlation between the percentage of γδ and vδ2 T cells between donor and recipient (Figure 1C).

As CMV reactivation impacts both the NK cell repertoire as well as the γδ T cell repertoire8,39, we analyzed the NK and γδ T cell repertoire 4 weeks after CMV reactivation (median day 41) compared it to NK and γδ T cells at day 60 of non reactivated patients (Figure 2). CMV reactivation showed no difference in NK cell percentages or in total γδ T cell percentages after MSD/MUD- or UCB-HCT. Both adaptive NK cells (defined as CD56+NKG2C+CD57+) as well as vδ1+ T cells were increased after CMV reactivation. In recipients of UCB-HCT but not of MSD/MUD-HCT, these increases were significant. This is most likely caused by a near absence of adaptive NK cells and vδ1+ T cells in CMV negative UCB-HCT recipients, as the magnitude of the adaptive NK cell response and vδ1+ T cell response is comparable between UCB-HCT and MSD/MUD-HCT after CMV reactivation.

Figure 2. NK cell and γδ T cell repertoire after CMV reactivation.

Figure 2

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Transplant samples were stratified for CMV reactivation. Samples were either analyzed 4 weeks after viral diagnosis (filled symbols) or 2-3 months after allo-HCT in CMV seronegative patients who did not show CMV reactivation (open symbols). UCB transplant recipients are shown in the upper panels (CMV reactivation n=13; no CMV reactivation n=14) and MSD/MUD transplant recipients in the lower panels (CMV reactivation n=14; no CMV reactivation n=15). Adaptive NK cells are defined as CD56+/CD3- NK cells that express CD57+NKG2C+. All cell fractions are presented as fraction of total lymphocytes. Statistics: Mann-Whitney test (* P<.05; ****p<0.0001).

Functionality of NK and vδ2 T cells responses can be addressed directly ex vivo on frozen samples

To best represent inherent activity without the confounding effects of cytokines, functional studies of NK and γδ T cells where performed directly on frozen PBMCs without further activation unless noted. NK cells derived from HD degranulate and produce cytokines upon incubation with a range of tumor cells after a 6-hour stimulation assay without the additional requirement for cytokines (Figure 3A). ZOL-treatment overnight did not affect NK cell mediated cytotoxicity in any of the four cell lines tested. Vδ1+ T cells (data not shown) and vδ2+ T cells (Figure 3B) show minimal tumor reactivity without further activation. However, when tumor cells were treated with ZOL overnight, vδ2+ T cells both show enhanced degranulation as well as cytokine production. For ZOL-treated K562, Raji and THP-1 targets, CD107a and cytokine levels are higher in vδ2+ cells as compared to NK cells. In contrast, ZOL-treated HL60 cells are not recognized by vδ2+ cells. This is in line with previous data that shows a variable degree of reactivity of Vγ9δ2 T cells against tumor cell lines which depends on the localization and distribution of Rho-B in in those cell lines21.

Figure 3. Zoledronate increases the functional response to tumors by vδ2 cells but not NK cells.

Figure 3

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Healthy donor samples with γδ T cells >1.5% of the lymphocyte gate were chosen for this analysis. Frozen PBMCs were thawed and rested overnight without cytokines. Functional analyses (CD107a degranulation and production of TNFα and IFNγ) were performed after a 6-hour incubation with the indicated tumor cell line with or without Zoledronate (20 uM) at an E:T ratio of 2:1. A) An example of the flow cytometry strategy for NK cells and γδ T cells is shown. B) Aggregate data is shown in panel B and presented as the mean ± SEM (n=6-14). Statistics: Mann-Whitney test (****p<0.0001).

Impact of IL-15 on NK and γδ T cell reactivity

IL-15 administration has been reported to enhance anti-leukemia effects40 after transplantation and high IL-15 levels at post-transplant day 7 correlate with reduced rates of cGVHD41. IL-15 has also been reported as potent stimulants of both NK42 and γδT cells27 which might partially explain the observed clinical effects. To dissect the impact of IL-15 on NK and γδ and αβT cells subsets frozen PBMCs of HD were thawed and rested overnight with or without IL-15 (10 ng/ml). IL-15 resulted in significantly increased NK cell mediated responses towards K562, Raji, HL60, and THP-1 (Figure 4A). The overall responses of vδ2 T cells were markedly lower as compared to NK cells. However, for vδ2 T cells, IL-15 resulted in a significant increase in degranulation and cytokine production for most tumor cell lines tested. Degranulation and cytokine production in vδ1+ T cells was lower as compared to vδ2+ T cells (Supplementary Figure 2). CD4 and CD8+ αβ T cells showed minimal function upon stimulation with IL-15 (data not shown).

Figure 4. Priming with IL-15 increases both NK and vδ2 function against tumors.

Figure 4

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A) Healthy donor samples with γδ T cells >1.5% of the lymphocyte gate were chosen for this analysis. Frozen PBMCs were thawed and rested overnight with or without IL-15 (10 ng/ml). The same functional analyses were performed by incubation with the indicated tumor cell line at an E:T ratio of 2:1. Aggregate functional data is shown as the mean (+SEM) as indicated (n= 6-14). B) Analysis of the IL2Rβ by flow cytometry in samples from healthy donors (left) or HCT recipients (right). HCT recipient samples were collected 2-3 months after MSD/MUD or UCB HCT. To allow direct comparison of of NK and γδ T cell repertoires, only samples with γδ T cell frequencies >1% of the lymphocyte gate were selected for evaluation (regardless of CMV reactivation). Analysis was only performed if > 150 events were available for assessment. Statistics: Mann-Whitney (*p<0.05; ** p<0.001; **** p<0.0001).

From these data it appears that NK cells are most potently stimulated by IL-15, but also vδ2+ T cells respond upon priming with IL-15. NK cells have significantly higher baseline reactivity towards tumor cell lines, but the fold-increase of the response upon IL-15 treatment is at least as high in vδ2+ T cells as in NK cells. The more abundant responsiveness of vδ2+ T cells as compared to vδ1+ T cells, as well as αβ T cells, may be explained by differential expression of the IL-15 receptor. The IL-15 receptor is one of the γc chain cytokine receptors, which has a unique IL-15Rα receptor and a shared IL-2/IL-15Rβ receptor43 (referred hereinafter as IL-2Rβ). IL-15Rα was under the limit of detection in our flow cytometric analysis, most likely explained by the fact that it is trans-presented by antigen presenting cells and monocytes. However, expression of IL-2Rβ could be quantified by flow cytometry. We therefore analyzed HD and patient samples 2-3 months post-HCT for expression of the IL-2Rβ (Figure 4B). We found that NK and vδ2+ cells had higher expression of IL-2Rβ as compared to vδ2 negative and conventional αβ T cells. Differential expression levels of the IL-2Rβ may therefore contribute to the differences in IL-15 induced priming of lymphocytes.

Role of NK and γδT and NK cells in ADCC

Monoclonal antibodies have been proposed as therapies after transplantation to improve further clinical outcome. To address the differential impact of NK cells and γδ T cell subsets on ADCC, relevant target cells were incubated with Rituximab or the 161533 TriKE32. We found that both Rituximab and the 161533 TriKE significantly induced NK and vδ2+ T cell function (Figure 5). Functionality upon incubation with Rituximab was comparable between NK cells and γδ T cells, whereas the 161533 TriKE appeared to result in somewhat higher NK cell responses as compared to γδ T cell responses (Figure 5A).

Figure 5. ADCC is mediated by NK, vδ2 and vδ1+ cells with wide ranges of CD16 expression.

Figure 5

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A) Frozen PBMCs of healthy donors were rested overnight without cytokines. Function was measured by incubation with the indicated tumor cell line with or without Rituximab (Rit, 10ug/ml) or a 161533 TriKE (30 nM) at an E:T ratio of 2:1. Only samples with at least 150 events were only included for analysis. Aggregate data are presented as the mean ± SEM (n=6-14). B) Representative sample of a healthy donor for CD16 expression on the indicated lymphocyte subsets. C) Aggregate data for CD16 expression in NK, γδT and αβT cells shown for healthy donors (left) or from patients 2-3 months after allo-HCT from MSD/MUD or UCB grafts (right). To allow direct comparison of of NK and γδ T cell repertoires, only samples with γδ T cell frequencies >1% of the lymphocyte gate were selected for evaluation (regardless of CMV reactivation). D) The correlation between CD16 expression and function is shown for degranulation after incubation with Raji cells + Rituximab. Statistics: A+C Mann-Whitney test: (* p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001); D Spearman non-parametric test.

We subsequently assessed CD16 expression on NK cells, as well as γδ T cells and αβ T cells in HD and patient samples. As expected, NK cells very brightly express CD16 (Figure 5B and C). CD8 and especially CD4 αβ T cells have low expression levels of CD16. When we analyzed CD16 expression on γδ T cells we found that CD16 expression on γδ T cells is higher as compared to αβ T cells. We also found that CD16 expression was very heterogeneous on γδ T cells. Within a single individual, CD16 expression can be very different between γδ T cell subsets (Figure 5B middle panel).

Given the heterogeneous expression of CD16 on γδ T cells, we sought to determine whether the level of CD16 expression correlated with the capacity of γδ T cells to perform ADCC. For vδ2+ T cells, we could indeed find a positive correlation of CD16 expression (both when defined in MFI (Figure 5D)) as well as defined in percentage (not shown) and function. For vδ1 T cells, however, such correlation was not present.

IL-15 and ZOL results synergistically prime patient derived NK and vδ2+ T cells

We subsequently tested how ZOL, IL-15 and the 161533 TriKE would affect NK and γδ T cell responses in patient-derived samples. We chose the THP-1 tumor model, as it can both initiate ZOL mediated vδ2 T cell responses and be targeted with the 161533 TriKE. To minimize the effect of immunosuppressive medication, we used patient samples obtained at 180 days post allo-HCT. NK and γδ T cell repertoires were comparable with respect to frequencies as well as to expression of surface molecules tested (CD16, TIGIT, PD-1, IL-2Rβ). To compare NK and γδ T cell responses, patients with γδ T cell frequencies >2% were selected for analysis. Here we show for THP-1 that addition of the 161533 TriKE induces degranulation as well as cytokine production in patient-derived NK cells (Figure 6). When NK cells are primed with IL-15, NK cells show significantly enhanced responses both directly and via ADCC. Patient-derived vδ2+ T cells show marked responses when tumor cells are treated with ZOL, which was very significantly increased after priming with IL-15.

Figure 6. The effect of IL15, Zoledronate and 161533 TriKE on patient derived NK and vδ2+ T cells.

Figure 6

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To allow direct comparison of the function of NK and γδ2+ γδ T cells, we selected HCT recipients from both graft sources (n=10) with γδ T cell frequencies of at least 2% in the lymphocyte gate in samples collected 6 months after HCT (regardless of CMV reactivation). Frozen PBMCs were thawed and rested overnight with or without IL15 (10 ng/ml). Only samples with at least 150 events were included in the analysis (n=10). NK and γδ2+ γδ T cells were incubated for 6 hours with THP-1 at an E:T ratio of 2:1. THP-1 cells were incubated with ZOL (20 uM) where indicted. The 161533 TriKE (30 nM) was added during the function assay where indicated. Statistical analysis was performed using a Mann-Whitney test (* p<0.05; ** p<0.01; ***p<.001; ****p<0.0001).

NK cells and γδ T cell subsets are differentially regulated by checkpoint molecules

Checkpoint inhibition is a major hurdle for strong anti-tumor reactivity but also protects patients after allo-HCT from severe GVHD. Checkpoint inhibitors might also be differentially regulated on different immune subsets. For instance vδ2- γδ T cells (including vδ1+ γδ T cells) have been reported to have a higher expression of IL-2Rβ, CD8α and the immune checkpoint receptors TIGIT (T cell Ig and ITIM domain) and LAG-3 (lymphocyte activation gene-3)44. On tumor-infiltrating conventional CD8+ αβ T cells, increased levels of TIGIT and LAG-3, and well as increased levels of ‘programmed cell death protein 1’ (PD-1) and ‘T-cell immunoglobulin and mucin-domain containing-3’ (TIM-3), can be found 45, 46. We therefore looked at expression of TIGIT as well as PD-1 on lymphocyte subsets of HD (Figure 7). We found that without IL-15 priming, overnight expression of TIGIT was higher in vδ1+ T cells as compared to NK cells and vδ2+ T cells (supplementary Figure 4). Overnight incubation with IL-15 significantly increased the expression of TIGIT in vδ1+ as well as vδ1-vδ2- double negative cells, but not in NK and vδ2+ cells. In contrast to TIGIT, PD-1 levels were comparable between lymphocyte subsets and also less affected by priming with IL-15 (Figure 7A and B right panels). After overnight priming with IL-15, we found a significant correlation between CD16 and TIGIT expression in vδ1+ T cells (Figure 7C), which was not observed in vδ2+ T cells (data not shown). Neither PD-1 nor IL2Rβ showed a correlation with CD16 expression in vδ1+ T cells (data not shown).

Figure 7. vδ1+ cells express high levels of TIGIT upon priming.

Figure 7

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Frozen PBMCs from healthy donors were rested overnight with (IL-15 Primed) or without (No IL-15) IL15 (10 ng/ml). Surface expression of TIGIT and PD-1 was assessed. Only samples with at least 150 events were included in the analysis. A) A representative example and B) aggregate data for TIGIT (left) and PD-1 (right) are shown for expression on NK, γδT and αβT cells. C) The correlation between CD16 expression and TIGIT/PD-1 expression on vδ1 cells. A Mann-Whitney test was used for the data in B (*p<0.05; ****p<0.0001) and Spearman non-parametric test for C.

Discussion

Our work is unique in that it simultaneously analyzes NK cells and γδ T cells after allo-HCT. In both populations, NK cell frequencies were higher as compared to HD. Recipients of UCB-HCT show marked NK cell IR, which is consistent with previous observations47. IR of γδ T cells, however, is strikingly different from NK cells. Vδ2+ T cells are virtually absent in recipients of UCB-HCT, as the γδ T cell repertoire in UCB has not yet undergone clonal differentiation induced by environmental factors, such as phosphoantigens48. We observed both in recipients of MSD/MUD-HCT as well as in HD, that a minority of individuals (around 30%) has higher vδ2 repertoires and a larger group (around 70%) have lower vδ2 repertoires. This may be compatible with a recently identified bimodal innate response towards a primary EBV infection in pediatric patients, in which half of the patients showed strong NK and γδ T cell responses, whereas the other half of patients showed NK cell responses accompanied by a feeble γδ T cell response49. Drivers of bimodal γδ T cell responses have not been identified. Also, whether these bimodal responses can be translated to other antigenic challenges remains to be established.

CMV infection or reactivation results in an increase in vδ1+ T cells, both in CMV+ HD as well as after MSD/MUD-HCT and UCB-HCT. Recently, next-generation sequencing of γδ TCR sequences revealed a clonal expansion of predominantly vδ1 or vδ3 TCRs in all CMV reactivated patients6. In contrast, by immune cell flow cytometry (ICF) we observed expansion of vδ1+ or vδ1-vδ2- T cells in only a fraction of patients. The study by Ravens et al6 does not provide quantitative information regarding the vδ1+ T cell response towards CMV. We therefore hypothesized that, in our cohort, either not all patients’ CMV+ vδ2- T cell clones undergo clonal expansion or clonal expansion remains under the limit of detection of ICF. It is also conceivable that CMV-induced vδ2- T cells may have migrated to peripheral organs at the time of analysis39. We also observed that an NK cell mediated CMV-response defined by the detection of ‘adaptive NK cells’ was present in a fraction of CMV-reactivated recipients. These data illustrate that multiple (innate) mechanisms can contribute to CMV responses in allo-HCT recipients39.

As NK cells are elevated in all MSD/MUD-HCT recipients and vδ2+ T cells in a significant proportion of patients, we explored a range of immunotherapeutic interventions to consider how both subsets might be targeted simultaneously. To evaluate endogenous function, we developed a functional assay to directly compare tumor responses by NK and γδ T cells derived from frozen PBMCs without the addition of cytokines or the requirement for expansion27.

Here we show that ZOL is a very potent enhancer of vδ2 mediated T cell responses, but that not every tumor cell line tested is equally susceptible. For example, ZOL-treated HL60 does not result in any vδ2 responsiveness. This confirms previous data, in which we have shown that ZOL mediated tumor recognition by vδ2+ T cells depends on a conformational change of BTN3A1. One of the requirements for such a conformational change is an altered cellular distribution of Rho-B. This associates with a Single Nucleotide Polymorphism (SNP), which can be found in 70% of normal individuals50. Although these data warrant validation in larger cohorts, it suggests that administration of bisphosphonates may be effective for a large proportion of individuals.

Administration of ZOL has recently been implemented shortly after αβ TCR / CD19-depleted haploidentical allo-HCT, an allo-HCT platform that favors IR of NK and γδ T cells. In this trial, ZOL administration resulted in activation of vδ2+ γδT cells, associating with improved clinical outcomes51. Ongoing challenges include determination of which factors contribute to beneficial outcomes of administration of bisphosphonates after allo-HCT. Success may depend on vδ2 IR, the vδ2 T cell repertoire of the donor, characteristics of the tumor cell including the mentioned SNP or the role of other (innate) immune cells. Although ZOL does not increase NK cell mediated immune responses directly ex vivo, indirect mechanisms by ZOL like stimulation of antigen-presenting cells52, alternations of the bone marrow niche by its effects on osteoclasts53 or by inhibition of Tregs 54 may contribute to NK cell mediated GVL responses in clinical scenarios.

In contrast to ZOL, we found that both IL-15 as well as antibody-based therapies simultaneously induced NK and vδ2+ T cell mediated tumor responses. γδ T cell mediated responses have been shown by others for trastuzumab 33, 34 or B cell specific antibodies like rituximab or CD19 specific bispecific antibodies35, 36. Here we show that the widely used monoclonal antibody Rituximab simultaneously induced robust NK and γδ T cell responses. A recently developed 161533 TriKE showed that CD33+ tumor cell lines HL60 and THP-1 are both targeted by NK cells and γδ T cells. We show that antibodies can be utilized to induce vδ2 mediated tumor responses for tumor types that were resistant to ZOL. When vδ2+ T cell responses were directly compared to NK cell responses, NK cell responses were superior. Not only were NK cell frequencies higher as compared to γδ T cell frequencies. Tumor functionality was also higher for most scenarios tested. These data illustrate that NK cells are the predominant ‘innate’ lymphocyte subset the first months after transplantation and that both IL-15 as well as antibodies can facilitate NK cell mediated tumor immune response. Vδ2+ T cells can contribute to these tumor immune responses by either simultaneous or alternative strategies.

Whereas NK and vδ2+ T cells can be stimulated by multiple strategies, vδ1+ showed not only a difference in IR, but also in responsiveness towards in vitro stimulation assays. In HD with a clearly detectable vδ1+ population, we were able to assess the response towards IL-15 and ADCC and found that vδ1+ showed inferior functionality as compared to vδ2+ T cells. Upon overnight stimulation with IL-15, vδ1 + T cells gained minimal tumor reactivity. This may be partially explained by a lower expression of IL2Rβ (part of the IL-15 receptor), although a significant proportion of vδ1+ T cells still have IL2Rβ expression levels comparable to that of NK cells. In addition, we did not find correlation between CD16 expression levels and ADCC. As vδ1+ T cells are mainly located outside PB, it is possible that vδ1+ T cells in peripheral tissue show differential activation profiles as compared to their circulating counterparts46.

For CD8+ αβ T cells, it has been established that persistent immune activation like tumor growth or chronic viral infection results in ‘exhaustion’. It remains less well established to what extend this can be expanded to γδ T cells. Recently, an epidermal γδ T cell population was characterized that expresses high levels of CD8α, IL2Rβ, TIGIT and LAG-344. We therefore tested expression of TIGIT on the γδ T cells and compared that to PD-1 (Figure 7). TIGIT (T cell Ig and ITIM domain) was recently shown to be expressed on Tregs 55, tumor infiltrating lymphocytes (TILs) 45, 56 and on epidermal γδ T cells 44. We found a strikingly increased expression of TIGIT in vδ2- subsets. Such expression was further increased upon stimulation with IL-15. PD-1 expression was comparable for all lymphocyte subsets. Within the vδ1+ T cell population, we found that TIGIT expression was increased in the CD8α+ vδ1+ T cell fraction (data not shown). CD8α has not only been described as being enriched on epidermal γδT cells44, it is also characterized as a signature of γδ T cells after CMV exposure as well as an essential co-receptor for leukemia-reactive vδ1 TCR17. In the functional analysis of CMV+ HD with significant vδ1 T cell populations, we observed a heterogenous expression of CD8α (Figure 6A). In addition, we found that that CD8α- vδ1+ cells trend to higher cytokine production upon incubation with antibodies, as compared to CD8α + T cells. When we analyzed TIGIT and CD16 expression in CD8α+ and CD8α- vδ1 T cells, we found in particular higher expression of TIGIT in CD8α + vδ1 cells which may explain why cytokine production is suppressed in CD8α+ vδ1+ T cells in our analyses.

We conclude that circulating vδ1+ T cells appear to have a phenotypic signature shared with epidermal γδ T cells and tumor-infiltrating lymphocytes and less with circulating vδ2+ T cells. To further elucidate the contribution of vδ1 T cell responses to tumor immunesurveillance, high throughput analysis of the tumor γδ T cell repertoire is warranted. This might also shed light on the observation that not all CD16 bright vδ1+ T cells show profound in vitro antibody mediated tumor reactivity in contrast to vδ2+ T cells and to αβ T cells in the context of a chronic viral infection 57.

Bisphosphonates, cytokines and various types of antibodies all have the capacity to induce innate immune responses post allo-HCT. Due to differential modes of action, combination therapy is likely to result in a synergistic innate tumor response as we have shown here for IL-15 and zoledronate. Successful implementation of these interventions may depend on stem cell source, graft manipulation and timing of the intervention therapy. Recently, αβ TCR graft depletion has been developed to decrease the incidence of GVHD. This platform results in rapid immune reconstitution of NK cells and γδ T cells 16, 58-60 without the requirement for immunosuppression. Future clinical trials exploring immune therapy after allo-HCT will not only elucidate therapeutic potential, but also provide innovative strategies to further understand mechanisms that drive innate immune surveillance.

Supplementary Material

Supplemental

Highlights.

  • NK cells and γδ T cells reconstitute early after allo-HCT

  • Reconstitution of vδ2+ cells depends on stem cell source and is less after cord blood transplant

  • Adaptive NK cells and Vδ1+ cells are enhanced by CMV reactivation

  • Bisphosphonates, IL-15, and targeting through CD16 enhance innate immunity after allo-HCT

Acknowledgments

This work was supported by the following NIH grants: P01 CA111412 (SC, MF, DAV, JSM), P01 CA65493 (JEW, MF, JSM), R35 CA197292 (MF, JSM), R01 HL122216 (MF, JSM).

Footnotes

COI Statement

There are no financial conflicts from any of the authors based on the work presented here.

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References

  • 1.Barrett AJ, Battiwalla M. Relapse after allogeneic stem cell transplantation. Expert review of hematology. 2010 Aug;3(4):429–441. doi: 10.1586/ehm.10.32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bosch M, Khan FM, Storek J. Immune reconstitution after hematopoietic cell transplantation. Current opinion in hematology. 2012 Jul;19(4):324–335. doi: 10.1097/MOH.0b013e328353bc7d. [DOI] [PubMed] [Google Scholar]
  • 3.Morvan MG, Lanier LL. NK cells and cancer: you can teach innate cells new tricks. Nature reviews Cancer. 2016 Jan;16(1):7–19. doi: 10.1038/nrc.2015.5. [DOI] [PubMed] [Google Scholar]
  • 4.Silva-Santos B, Serre K, Norell H. gammadelta T cells in cancer. Nature reviews Immunology. 2015 Nov;15(11):683–691. doi: 10.1038/nri3904. [DOI] [PubMed] [Google Scholar]
  • 5.Ullah MA, Hill GR, Tey SK. Functional Reconstitution of Natural Killer Cells in Allogeneic Hematopoietic Stem Cell Transplantation. Frontiers in immunology. 2016;7:144. doi: 10.3389/fimmu.2016.00144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ravens S, Schultze-Florey C, Raha S, Sandrock I, Drenker M, Oberdorfer L, et al. Human gammadelta T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection. Nature immunology. 2017 Feb;:20. doi: 10.1038/ni.3686. [DOI] [PubMed] [Google Scholar]
  • 7.Scheper W, Grunder C, Straetemans T, Sebestyen Z, Kuball J. Hunting for clinical translation with innate-like immune cells and their receptors. Leukemia. 2014 Jun;28(6):1181–1190. doi: 10.1038/leu.2013.378. [DOI] [PubMed] [Google Scholar]
  • 8.de Witte MA, Kuball J, Miller JS. NK Cells and γδT Cells for Relapse Protection after Allogeneic Hematopoietic Cell Transplantation (HCT) Curr Stem Cell Rep. 2017;3(4):301–311. doi: 10.1007/s40778-017-0106-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Boeckh M, Nichols WG, Papanicolaou G, Rubin R, Wingard JR, Zaia J. Cytomegalovirus in hematopoietic stem cell transplant recipients: Current status, known challenges, and future strategies. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 2003 Sep;9(9):543–558. doi: 10.1016/s1083-8791(03)00287-8. [DOI] [PubMed] [Google Scholar]
  • 10.Foley B, Cooley S, Verneris MR, Pitt M, Curtsinger J, Luo X, et al. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood. 2012 Mar 15;119(11):2665–2674. doi: 10.1182/blood-2011-10-386995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cichocki F, Cooley S, Davis Z, DeFor TE, Schlums H, Zhang B, et al. CD56dimCD57+NKG2C+ NK cell expansion is associated with reduced leukemia relapse after reduced intensity HCT. Leukemia. 2016 Feb;30(2):456–463. doi: 10.1038/leu.2015.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Elmaagacli AH, Steckel NK, Koldehoff M, Hegerfeldt Y, Trenschel R, Ditschkowski M, et al. Early human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in acute myeloid leukemia patients. Blood. 2011 Aug 4;118(5):1402–1412. doi: 10.1182/blood-2010-08-304121. [DOI] [PubMed] [Google Scholar]
  • 13.Green ML, Leisenring WM, Xie H, Walter RB, Mielcarek M, Sandmaier BM, et al. CMV reactivation after allogeneic HCT and relapse risk: evidence for early protection in acute myeloid leukemia. Blood. 2013 Aug 15;122(7):1316–1324. doi: 10.1182/blood-2013-02-487074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Dechanet J, Merville P, Lim A, Retiere C, Pitard V, Lafarge X, et al. Implication of gammadelta T cells in the human immune response to cytomegalovirus. The Journal of clinical investigation. 1999 May 15;103(10):1437–1449. doi: 10.1172/JCI5409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Knight A, Madrigal AJ, Grace S, Sivakumaran J, Kottaridis P, Mackinnon S, et al. The role of Vdelta2-negative gammadelta T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation. Blood. 2010 Sep 23;116(12):2164–2172. doi: 10.1182/blood-2010-01-255166. [DOI] [PubMed] [Google Scholar]
  • 16.Airoldi I, Bertaina A, Prigione I, Zorzoli A, Pagliara D, Cocco C, et al. gammadelta T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-alphabeta+/CD19+ lymphocytes. Blood. 2015 Apr 9;125(15):2349–2358. doi: 10.1182/blood-2014-09-599423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Scheper W, van Dorp S, Kersting S, Pietersma F, Lindemans C, Hol S, et al. gammadeltaT cells elicited by CMV reactivation after allo-SCT cross-recognize CMV and leukemia. Leukemia. 2013 Jun;27(6):1328–1338. doi: 10.1038/leu.2012.374. [DOI] [PubMed] [Google Scholar]
  • 18.Godder KT, Henslee-Downey PJ, Mehta J, Park BS, Chiang KY, Abhyankar S, et al. Long term disease-free survival in acute leukemia patients recovering with increased gammadelta T cells after partially mismatched related donor bone marrow transplantation. Bone marrow transplantation. 2007 Jun;39(12):751–757. doi: 10.1038/sj.bmt.1705650. [DOI] [PubMed] [Google Scholar]
  • 19.Gober HJ, Kistowska M, Angman L, Jeno P, Mori L, De Libero G. Human T cell receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med. 2003 Jan 20;197(2):163–168. doi: 10.1084/jem.20021500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Harly C, Guillaume Y, Nedellec S, Peigne CM, Monkkonen H, Monkkonen J, et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human gammadelta T-cell subset. Blood. 2012 Sep 13;120(11):2269–2279. doi: 10.1182/blood-2012-05-430470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Sebestyen Z, Scheper W, Vyborova A, Gu S, Rychnavska Z, Schiffler M, et al. RhoB Mediates Phosphoantigen Recognition by Vgamma9Vdelta2 T Cell Receptor. Cell Rep. 2016 May 31;15(9):1973–1985. doi: 10.1016/j.celrep.2016.04.081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rautela J, Huntington ND. IL-15 signaling in NK cell cancer immunotherapy. Current opinion in immunology. 2016 Nov 08;44:1–6. doi: 10.1016/j.coi.2016.10.004. [DOI] [PubMed] [Google Scholar]
  • 23.Bachanova V, Cooley S, Defor TE, Verneris MR, Zhang B, McKenna DH, et al. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood. 2014 Jun 19;123(25):3855–3863. doi: 10.1182/blood-2013-10-532531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, Embers M, et al. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med. 2000 Mar 06;191(5):771–780. doi: 10.1084/jem.191.5.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Garcia VE, Jullien D, Song M, Uyemura K, Shuai K, Morita CT, et al. IL-15 enhances the response of human gamma delta T cells to nonpeptide [correction of nonpetide] microbial antigens. J Immunol. 1998 May 1;160(9):4322–4329. [PubMed] [Google Scholar]
  • 26.Cairo C, Sagnia B, Cappelli G, Colizzi V, Leke RG, Leke RJ, et al. Human cord blood gammadelta T cells expressing public Vgamma2 chains dominate the response to bisphosphonate plus interleukin-15. Immunology. 2013 Apr;138(4):346–360. doi: 10.1111/imm.12039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ribot JC, Ribeiro ST, Correia DV, Sousa AE, Silva-Santos B. Human gammadelta thymocytes are functionally immature and differentiate into cytotoxic type 1 effector T cells upon IL-2/IL-15 signaling. J Immunol. 2014 Mar 1;192(5):2237–2243. doi: 10.4049/jimmunol.1303119. [DOI] [PubMed] [Google Scholar]
  • 28.Conlon KC, Lugli E, Welles HC, Rosenberg SA, Fojo AT, Morris JC, et al. Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2015 Jan 01;33(1):74–82. doi: 10.1200/JCO.2014.57.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Braakman E, van de Winkel JG, van Krimpen BA, Jansze M, Bolhuis RL. CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes. Cell Immunol. 1992 Aug;143(1):97–107. doi: 10.1016/0008-8749(92)90008-d. [DOI] [PubMed] [Google Scholar]
  • 30.Weiner GJ. Rituximab: mechanism of action. Semin Hematol. 2010 Apr;47(2):115–123. doi: 10.1053/j.seminhematol.2010.01.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Felices M, Lenvik TR, Davis ZB, Miller JS, Vallera DA. Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells. Methods Mol Biol. 2016;1441:333–346. doi: 10.1007/978-1-4939-3684-7_28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Vallera DA, Felices M, McElmurry R, McCullar V, Zhou X, Schmohl JU, et al. IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing Persistence, In Vivo Expansion, and Enhanced Function. Clinical cancer research : an official journal of the American Association for Cancer Research. 2016 Jul 15;22(14):3440–3450. doi: 10.1158/1078-0432.CCR-15-2710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Capietto AH, Martinet L, Fournie JJ. Stimulated gammadelta T cells increase the in vivo efficacy of trastuzumab in HER-2+ breast cancer. J Immunol. 2011 Jul 15;187(2):1031–1038. doi: 10.4049/jimmunol.1100681. [DOI] [PubMed] [Google Scholar]
  • 34.Tokuyama H, Hagi T, Mattarollo SR, Morley J, Wang Q, So HF, et al. V gamma 9 V delta 2 T cell cytotoxicity against tumor cells is enhanced by monoclonal antibody drugs--rituximab and trastuzumab. Int J Cancer. 2008 Jun 01;122(11):2526–2534. doi: 10.1002/ijc.23365. [DOI] [PubMed] [Google Scholar]
  • 35.Schiller CB, Braciak TA, Fenn NC, Seidel UJ, Roskopf CC, Wildenhain S, et al. CD19-specific triplebody SPM-1 engages NK and gammadelta T cells for rapid and efficient lysis of malignant B-lymphoid cells. Oncotarget. 2016 Nov 04; doi: 10.18632/oncotarget.13110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Seidel UJ, Vogt F, Grosse-Hovest L, Jung G, Handgretinger R, Lang P. gammadelta T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay. Frontiers in immunology. 2014;5:618. doi: 10.3389/fimmu.2014.00618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.de Koning C, Plantinga M, Besseling P, Boelens JJ, Nierkens S. Immune Reconstitution after Allogeneic Hematopoietic Cell Transplantation in Children. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 2016 Feb;22(2):195–206. doi: 10.1016/j.bbmt.2015.08.028. [DOI] [PubMed] [Google Scholar]
  • 38.Scheper W, Sebestyen Z, Kuball J. Cancer Immunotherapy Using gammadeltaT Cells: Dealing with Diversity. Frontiers in immunology. 2014;5:601. doi: 10.3389/fimmu.2014.00601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Khairallah C, Dechanet-Merville J, Capone M. gammadelta T Cell-Mediated Immunity to Cytomegalovirus Infection. Frontiers in immunology. 2017;8:105. doi: 10.3389/fimmu.2017.00105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Sauter CT, Bailey CP, Panis MM, Biswas CS, Budak-Alpdogan T, Durham A, et al. Interleukin-15 administration increases graft-versus-tumor activity in recipients of haploidentical hematopoietic SCT. Bone marrow transplantation. 2013 Sep;48(9):1237–1242. doi: 10.1038/bmt.2013.47. [DOI] [PubMed] [Google Scholar]
  • 41.Pratt LM, Liu Y, Ugarte-Torres A, Hoegh-Petersen M, Podgorny PJ, Lyon AW, et al. IL15 levels on day 7 after hematopoietic cell transplantation predict chronic GVHD. Bone marrow transplantation. 2013 May;48(5):722–728. doi: 10.1038/bmt.2012.210. [DOI] [PubMed] [Google Scholar]
  • 42.Alpdogan O, Eng JM, Muriglan SJ, Willis LM, Hubbard VM, Tjoe KH, et al. Interleukin-15 enhances immune reconstitution after allogeneic bone marrow transplantation. Blood. 2005 Jan 15;105(2):865–873. doi: 10.1182/blood-2003-09-3344. [DOI] [PubMed] [Google Scholar]
  • 43.Rochman Y, Spolski R, Leonard WJ. New insights into the regulation of T cells by gamma(c) family cytokines. Nature reviews Immunology. 2009 Jul;9(7):480–490. doi: 10.1038/nri2580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Di Marco Barros R, Roberts NA, Dart RJ, Vantourout P, Jandke A, Nussbaumer O, et al. Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific gammadelta T Cell Compartments. Cell. 2016 Sep 22;167(1):203–218. e217. doi: 10.1016/j.cell.2016.08.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Chauvin JM, Pagliano O, Fourcade J, Sun Z, Wang H, Sander C, et al. TIGIT and PD-1 impair tumor antigen-specific CD8(+) T cells in melanoma patients. The Journal of clinical investigation. 2015 May;125(5):2046–2058. doi: 10.1172/JCI80445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Singer M, Wang C, Cong L, Marjanovic ND, Kowalczyk MS, Zhang H, et al. A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells. Cell. 2016 Sep 08;166(6):1500–1511. e1509. doi: 10.1016/j.cell.2016.08.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Szabolcs P, Niedzwiecki D. Immune reconstitution in children after unrelated cord blood transplantation. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 2008 Jan;14(1 Suppl 1):66–72. doi: 10.1016/j.bbmt.2007.10.016. [DOI] [PubMed] [Google Scholar]
  • 48.Morita CT, Parker CM, Brenner MB, Band H. TCR usage and functional capabilities of human gamma delta T cells at birth. J Immunol. 1994 Nov 01;153(9):3979–3988. [PubMed] [Google Scholar]
  • 49.Djaoud Z, Guethlein LA, Horowitz A, Azzi T, Nemat-Gorgani N, Olive D, et al. Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and gammadelta T cells. J Exp Med. 2017 May 03; doi: 10.1084/jem.20161017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Sebestyen Z, Scheper W, Vyborova A, Gu S, Rychnavska Z, Schiffler M, et al. RhoB Mediates Phosphoantigen Recognition by Vgamma9Vdelta2 T Cell Receptor. Cell Rep. 2016 May 19; doi: 10.1016/j.celrep.2016.04.081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Bertaina A, Zorzoli A, Petretto A, Barbarito G, Inglese E, Merli P, et al. Zoledronic acid boosts gammadelta T-cell activity in children receiving alphabeta+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor. Oncoimmunology. 2017;6(2):e1216291. doi: 10.1080/2162402X.2016.1216291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Maniar A, Zhang X, Lin W, Gastman BR, Pauza CD, Strome SE, et al. Human gammadelta T lymphocytes induce robust NK cell-mediated antitumor cytotoxicity through CD137 engagement. Blood. 2010 Sep 9;116(10):1726–1733. doi: 10.1182/blood-2009-07-234211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.An G, Acharya C, Feng X, Wen K, Zhong M, Zhang L, et al. Osteoclasts promote immune suppressive microenvironment in multiple myeloma: therapeutic implication. Blood. 2016 Sep 22;128(12):1590–1603. doi: 10.1182/blood-2016-03-707547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Sarhan D, Leijonhufvud C, Murray S, Witt K, Seitz C, Wallerius M, et al. Zoledronic acid inhibits NFAT and IL-2 signaling pathways in regulatory T cells and diminishes their suppressive function in patients with metastatic cancer. Oncoimmunology. 2017 doi: 10.1080/2162402X.2017.1338238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Joller N, Lozano E, Burkett PR, Patel B, Xiao S, Zhu C, et al. Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses. Immunity. 2014 Apr 17;40(4):569–581. doi: 10.1016/j.immuni.2014.02.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector function. Cancer cell. 2014 Dec 08;26(6):923–937. doi: 10.1016/j.ccell.2014.10.018. [DOI] [PubMed] [Google Scholar]
  • 57.Bjorkstrom NK, Gonzalez VD, Malmberg KJ, Falconer K, Alaeus A, Nowak G, et al. Elevated numbers of Fc gamma RIIIA+ (CD16+) effector CD8 T cells with NK cell-like function in chronic hepatitis C virus infection. J Immunol. 2008 Sep 15;181(6):4219–4228. doi: 10.4049/jimmunol.181.6.4219. [DOI] [PubMed] [Google Scholar]
  • 58.Locatelli F, Merli P, Pagliara D, Li Pira G, Falco M, Pende D, et al. Outcome of children with acute leukemia given HLA-haploidentical HSCT after alphabeta T-cell and B-cell depletion. Blood. 2017 Jun 06; doi: 10.1182/blood-2017-04-779769. [DOI] [PubMed] [Google Scholar]
  • 59.Zvyagin IV, Mamedov IZ, Tatarinova OV, Komech EA, Kurnikova EE, Boyakova EV, et al. Tracking T-cell immune reconstitution after TCRalphabeta/CD19-depleted hematopoietic cells transplantation in children. Leukemia. 2016 Dec 09; doi: 10.1038/leu.2016.321. [DOI] [PubMed] [Google Scholar]
  • 60.Lang P, Feuchtinger T, Teltschik HM, Schwinger W, Schlegel P, Pfeiffer M, et al. Improved immune recovery after transplantation of TCRalphabeta/CD19-depleted allografts from haploidentical donors in pediatric patients. Bone marrow transplantation. 2015 Jun;50(Suppl 2):S6–10. doi: 10.1038/bmt.2015.87. [DOI] [PubMed] [Google Scholar]

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